Endothelial Function in Patients with Sickle Cell Anemia During and After Sickle Cell Crises

Background:Prior studies have demonstrated increased adherence of sickle cell erythrocytes to vascular endothelial cells. While decreased production of nitric oxide and increased production of adhesion molecules have been implicated in this pathophysiology, the relative contribution of these mechanisms during acute sickle cell crises as compared to steady state conditions have not been elucidated.Methods and results: We studied 10 consecutive young adult patients presenting with a sickle cell crisis. Endothelial function was evaluated by a non-invasive brachial artery shear stress method. Serum levels of adhesion molecules were obtained during the crisis. Both brachial artery responsiveness and serum levels of adhesion molecules were then repeated at steady state. Ten age and gender matched volunteers served as a control group. Impaired endothelial function and impaired endothelium-independent vasodilatation were observed in all sickle cell patients during both steady state and during crisis. Flow mediated dilation (FMD)% was 3.25 ± 2.76% during crisis, 4.57 ± 4.11 at steady state, compared with the control group FMD of 11.64 ± 7.69% (p < 0.001). Flow independent dilation was 10.35 ± 11.3% during crisis, 10.03 ± 6.52% at steady state, compared with control group FID of 24.17 ± 11.87% (p < 0.001). Levels of cell adhesion molecules and markers of inflammation were increased in sickle cell crisis patients compared with the control group: sCD40 ligand levels during the acute crisis were over twice the level of normal matched volunteers (p = 0.02), and similarly significant increases were seen for E-selectin (p = 0.008), ICAM-1 (p = 0.037) and VCAM-1 levels (p = 0.01). The levels of each of these biomarkers was not significantly increased during acute crises as compared to patients’ recovery state.Conclusions: Sickle cell anemia patients have severe systemic endothelial dysfunction as demonstrated by both brachial artery assessment and increased serum levels of adhesion molecules. These abnormalities characterize not only the sickle cell crisis but also the steady state pathophysiology of sickle cell anemia.     

Levels of soluble endothelium-derived adhesion molecules in patients with sickle cell disease are associated with pulmonary hypertension, organ dysfunction, and mortality

Endothelial cell adhesion molecules orchestrate the recruitment and binding of inflammatory cells to vascular endothelium. With endothelial dysfunction and vascular injury, the levels of endothelial bound and soluble adhesion molecules increase. Such expression is modulated by nitric oxide (NO), and in patients with sickle cell disease (SCD), these levels are inversely associated with measures of NO bioavailability. To further evaluate the role of endothelial dysfunction in a population study of SCD, we have measured the levels of soluble endothelium-derived adhesion molecules in the plasma specimens of 160 adult patients with SCD during steady state. Consistent with a link between endothelial dysfunction and end-organ disease, we found that higher levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) were associated with markers indicating renal dysfunction and hepatic impairment. Analysis of soluble intercellular cell adhesion molecule-1 (sICAM-1), sE-selectin and sP-selectin levels indicated partially overlapping associations with sVCAM-1, with an additional association with inflammatory stress and triglyceride levels. Importantly, increased soluble adhesion molecule expression correlated with severity of pulmonary hypertension, a clinical manifestation of endothelial dysfunction. Soluble VCAM-1, ICAM-1, and E-selectin were independently associated with the risk of mortality in this cohort. Our data are consistent with steady state levels of soluble adhesion molecules as markers of pulmonary hypertension and risk of death.     

Modulation by high glucose of adhesion molecule expression in cultured endothelial cells

Summary We evaluated the influence of high ambient glucose on cellular expression of adhesion molecules, known to mediate endothelial interaction of leucocytes and monocytes. Paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were studied by fluorescence activated cell sorter analysis after exposure to 30 vs 5 mmol/l glucose. Incubation of HUVECs for 24 h in 30 mmol/l glucose increased ICAM-1 (intercellular adhesion molecule-1; 116.4±16.9% of control, p 0.05), but not PECAM (platelet endothelial cell adhesion molecule) expression, compared to cultures kept in 5 mmol/l glucose. Long-term exposure (13±1 days) of HUVECs to 30 mmol/l glucose increased expression of ICAM-1 to 122.5±32.2% (p<0.002) and reduced that of PECAM to 86.9±21.3% vs the respective control culture in 5 mmol/l glucose (p<0.02). Stimulation of confluent HUVECs, kept in 30 vs 5 mmol/l glucose for 13±1 days, with 20 U/ ml interleukin-1 for 24 h (ICAM-1) and 4 h (endothelial leukocyte adhesion molecule 1) resulted in reduced ICAM-1 (84.8±27.0%, p<0.05) and endothelial leukocyte adhesion molecule-1 (87.6±22.4%, p<0.05) expression vs control cells, while that of PECAM (t: 24 h) and vascular cell adhesion molecule-1 (t: 16 h) remained unchanged. In conclusion, it appears that differences in expression of adhesion molecules on HUVECs in response to high glucose reflects endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.Abbreviations HUVECs Human umbilical vein endothelial cells - ICAM-1 intercellular adhesion molecule-1 - PECAM platelet endothelial cell adhesion molecule - ELAM-1 endothelial leukocyte adhesion molecule-1 - VCAM-1 vascular cell adhesion molecule-1 - IL-1 interleukin-1 - FACS fluorescence activated cell sorter - FCS fetal calf serum - PBS phosphate buffered saline     

Abnormalities of coagulation and fibrinolysis in homozygous sickle cell disease

Abnormalities of coagulation and fibrinolysis were studied in a group of 28 children and young adults with homozygous sickle cell disease (SCD), either in the steady state (n = 12) or during painful crisis (n = 16). Coagulation was explored by standard clotting tests and by measurement of prothrombin complex factors, factor VIII (VIII:C) and antithrombin III (ATIII), protein C (PC) and protein S (PS) activities, while fibrinolytic potential was evaluated using D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) assays. In SCD patients, thrombin time (TT) was constantly shortened, both in the steady state (ratio to control 0.83 ± 0.08, p < 0.0001) and in crisis (0.76 ± 0.06, p < 0.0001). Mean levels of prothrombin complex were similar in asymptomatic patients to those in controls, but were significantly decreased during sickle cell crisis (p < 0.05 for factor V and p < 0.0001 for factors II,VII and X). Factor VIII:C was significantly increased, both in the steady state (207 ± 35%, p < 0.0001) and during crisis (208 ± 34 %, p < 0,0001). PS activity was reduced in the steady state (81 ± 12%, p < 0.01) and further diminished in crisis (68.5 ± 27.5%, p < 0.001), while D-dimers were significantly elevated during sickle cell crisis (1028 ± 675 ng/ml, p < 0.001). In all SCD patients, baseline levels of t-PA antigen were comparable to those in controls, whereas concentrations of PAI-1 antigen were significantly increased, either in the steady state (89.7 ± 26.3 ng/ml, p < 0.0001) or in crisis (75.0 ± 24.8 ng/ml, p < 0.0001). These results provide evidence for the presence of circulating activated clotting factors in SCD and for an imbalance of the profibrinolytic and antifibrinolytic systems most likely due to increased PAI-1 levels.     

Sickle cell acute chest syndrome: pathogenesis and rationale for treatment.

Acute chest syndrome (ACS) is a leading cause of death in sickle cell disease (SCD). Our previous work showed that hypoxia enhances the ability of sickle erythrocytes to adhere to human microvessel endothelium via interaction between very late activation antigen-4 (VLA4) expressed on sickle erythrocytes and the endothelial adhesion molecule vascular cell adhesion molecule-1 (VCAM-1). Additionally, hypoxia has been shown to decrease the production of nitric oxide (NO) which inhibits VCAM-1 upregulation. Based on these observations, we hypothesize that during ACS, the rapidly progressive clinical course that can occur is caused by initial hypoxia-induced pulmonary endothelial VCAM-1 upregulation that is not counterbalanced by production of cytoprotective mediators, including NO, resulting in intrapulmonary adhesion. We assessed plasma NO metabolites and soluble VCAM-1 in 36 patients with SCD and 23 age-matched controls. Patients with SCD were evaluated at baseline (n = 36), in vaso-occlusive crisis (VOC; n = 12), and during ACS (n = 7). We observed marked upregulation of VCAM-1 during ACS (1,290 +/- 451 ng per mL; mean +/- 1 SD) with values significantly higher than controls (P <.0001) or patients either in steady state or VOC (P <. 01). NO metabolites were concomitantly decreased during ACS (9.2 +/- 1.5 nmol/mL) with values lower than controls (22.2 +/- 5.5), patients during steady state (21.4 +/- 5.5), or VOC (14.2 +/- 1.2) (P <.0001). Additionally, the ratio of soluble VCAM-1 to NO metabolites during ACS (132.9 +/- 46.5) was significantly higher when compared with controls (P <.0001) or patients either in steady state or VOC (P <.0001). Although hypoxia enhanced in vitro sickle erythrocyte-pulmonary microvessel adhesion, NO donors inhibited this process with concomitant inhibition of VCAM-1. We suggest that in ACS there is pathologic over expression of endothelial VCAM-1. Our investigations also provide a rationale for the therapeutic use in ACS of cytoprotective modulators including NO and dexamethasone, which potentially exert their efficacy by an inhibitory effect on VCAM-1 and concomitant inhibition of sickle erythrocyte-endothelial adhesion.     

Pulmonary arterial hypertension and left-sided heart disease in sickle cell disease: clinical characteristics and association with soluble adhesion molecule expression

Pulmonary hypertension (PH), a risk factor for mortality in sickle cell disease (SCD), has pathologic features of both pulmonary arterial hypertension (PAH) and PH related to left-sided heart disease (LHD) suggesting a link between these two entities. We hypothesized that both are characterized by endothelial dysfunction and increased adhesion molecule expression. SCD patients and normal volunteers underwent a screening questionnaire, echocardiogram, and blood donation for preparation of platelet-poor plasma. PAH was defined as a tricuspid regurgitant jet (TRJ) velocity > or =2.5 m/sec and/or the presence of isolated right ventricular hypertrophy or decreased systolic function. LHD was defined as either left-sided systolic/diastolic dysfunction or significant valvular disease. Plasma vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P- and E-selectin, nitric oxide (NO(x)), erythropoietin, and vascular endothelial growth factor (VEGF) levels were assayed by enzyme-linked immunoassay. Forty-three percent of sickle cell anemia (HbSS) and 28% of hemoglobin SC disease (HbSC) disease patients had PAH. Additionally, 10-15% of SCD patients had LHD. VCAM-1 levels were significantly increased in HbSS patients compared with HbSC patients and normal volunteers. VCAM-1 and P-selectin levels correlated positively with TRJ velocity in HbSS patients (r = 0.45, P = 0.03, r = 0.2, P = 0.05, respectively). ICAM-1, E-selectin, NO(x), erythropoietin, and VEGF levels were similar across subject groups. PH is common in SCD and, at times, due to LHD. Increased VCAM-1 and P-selectin expression was associated with TRJ elevation regardless of etiology suggesting a similar effect on endothelial gene expression and possibly providing a pathologic link between PAH and PH related to LHD in 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.     

Association of soluble fms-like tyrosine kinase-1 with pulmonary hypertension and haemolysis in sickle cell disease

The pathophysiology of pulmonary hypertension (PHT) in sickle cell disease (SCD) is probably multifactorial. Soluble fms-like tyrosine kinase-1 (sFLT-1) is a member of the vascular endothelial growth factor receptor (VEGFR) family. By adhering to and inhibiting VEGF and placenta growth factor, it induces endothelial dysfunction. We sought to evaluate the association of sFLT-1 with clinical complications of SCD. We confirmed that sFLT-1 was significantly elevated in SCD patients compared to healthy, race-matched control subjects. The level of sFLT-1 was significantly higher in patients with PHT, but no association was observed between sFLT-1 and the frequency of acute pain episodes or history of acute chest syndrome. sFLT-1 was correlated with various measures of haemolysis, erythropoietin and soluble vascular cell adhesion molecule-1. By inducing endothelial dysfunction, sFLT-1 may contribute to the pathogenesis of SCD-associated PHT, although this effect does not appear to be independent of haemolysis.     

Blood pressure disturbances and endothelial dysfunction markers in children and adolescents with type 1 diabetes

Objective: Being the earliest step on the way to atherosclerosis, endothelial dysfunction is particularly escalated in diabetes. This study aimed at assessing endothelial dysfunction and blood pressure disturbances in young patients with type 1 diabetes mellitus (T1DM) and defining their interrelations. Methods: The study group comprised 52 children and adolescents aged 14.07 ± 3.03 years, with T1DM duration 5.13 ± 2.18 years. 20 healthy controls with similar age and sex distribution were included. Chosen serum biochemical markers of endothelial damage: intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), sE-selectin, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) as well as ambulatory blood pressure monitoring (ABPM) were performed in all subjects. Results: Patients with T1DM displayed significantly higher concentrations of chosen markers of endothelial dysfunction compared to controls (sVCAM-1 (ng/ml): 951.56 ± 330.68 vs. 710.35 ± 162.12, TNF-α (pg/ml): 16.63 ± 8.32 vs. 9.41 ± 4.23, IL-6 (pg/ml): 3.38 ± 1.31 vs. 2.45 ± 0.81; p < 0.05). Within the study group subjects with an abnormal ABPM reading had significantly higher concentrations of sE-selectin compared with subjects with normal ABPM (in ng/ml: 45.71 ± 15.63 vs. 32.42 ± 11.95; p < 0.01). The study revealed a significant positive correlation between sE-selectin and systolic as well as diastolic pressure loads during the day period (respectively: r = 0.46, r = 0.60; p < 0.01). Conclusions: Endothelium dysfunction may be present early in the course of T1DM in children and adolescents. It seems to be related with blood pressure disturbances which highlights the need to intensify treatment in this group of patients.     

Marked elevation of vascular endothelial growth factor and basic fibroblast growth factor in pericardial fluid of patients with angina pectoris

Although we reported that basic fibroblast growth factor (bFGF) levels in pericardial fluid of patients with unstable angina are apparently increased, it was unclear whether vascular endothelial growth factor (VEGF) is also increased in patients with myocardial ischemia. Using an enzyme-linked immunosorbent assay, we measured the concentrations of VEGF and bFGF in pericardial fluid of 51 patients with open heart surgery. Patients were divided into group A (n=10) with class III unstable angina (Braunwald's classification), group B (n=24) with class I or II unstable angina or stable angina and group C (n=17) with non-ischemic heart disease. The VEGF level in pericardial fluid in group A was 83±7 pg/ml, being significantly (p<0.001) higher than the 27±3 pg/ml in group B and the 28±5 pg/ml in group C. The concentrations of bFGF in pericardial fluid in groups A and B were 1461±579 and 1224±161 pg/ml, respectively, significantly (p<0.05) higher than the 292±97 pg/ml in group C. The level of VEGF in pericardial fluid was increased only in patients with severe rest angina within 2 days before emergency coronary artery bypass graft surgery (CABG), while bFGF was increased in all patients undergoing CABG for coronary artery disease. Thus VEGF and bFGF may play important roles in mediating collateral growth in humans.     

Endothelial activation and inflammation biomarkers in children and adolescents with sickle cell disease

Sickle cell disease pathogenesis is a complex interplay of multiple factors associated with vascular endothelial activation, intense oxidative stress, and increased sickle cell adhesion. The aim of this study was to determine and compare three panels of plasma circulating biomarkers at ‘steady state’ and during veno-occlusive crises (VOC) in a cohort of children and adolescents with SCD and healthy controls. The following biomarkers were assessed: acute phase reactants, endothelial factors, and adhesion molecules. Forty-one SCD pediatric patients and 28 healthy children were enrolled. Patients at ‘steady state’ presented significantly elevated plasma levels of endothelin-1 (ET-1), soluble-VCAM-1 (sVCAM-1), soluble P-selectin (sP-selectin), and d-dimers compared to the control group. ET-1, sP-selectin, platelet-derived growth factor (PDGF), von Willebrand factor (vWf), d-dimers, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) seems to represent additional, but not independent, prognostic markers of VOC crisis. Elevated plasma levels of sP-selectin, ET-1, and sVCAM-1 were associated with VOC frequency. The present study provides preliminary evidence of a possible association between these biomarkers and the endothelial activation at steady state and VOC in childhood SCD. Further prospective studies are required to confirm the potential independent prognostic value of these markers in different stages of pediatric SCD.     

VLA‐4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow

Very Late Antigen‐4 (VLA‐4, α4β1‐integrin, ITGA4) orchestrates cell‐cell and cell‐endothelium adhesion. Given the proposed role of VLA‐4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA‐4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA‐4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA‐4, while VLA‐4 staining of non‐SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA‐4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM‐1) was blocked by natalizumab in a dose‐dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF‐α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA‐4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM‐1 and that the VLA‐4 adhesion to VCAM‐1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA‐4 blockade may be beneficial in sickle cell disease.     

Biomarkers of inflammation, growth factor, and coagulation activation in patients with sickle cell disease

Acute painful crisis is a common sequela that can cause significant morbidity and negatively impact the quality of life of patients with sickle cell disease (SCD). Plasma levels of several chemokines and cytokines including tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), and interferon γ (IFN-γ) in patients with SCD showed a distinct and statistically significant rise either during painful crisis or at steady state. Plasma levels of various growth factors, including human vascular endothelial growth factor (VEGF), human basic fibroblast growth factor (FGF), and human hepatocyte growth factor (HGF), showed a sustained 2- to 3-fold increase either during painful crisis or at steady state in patients with SCD. Furthermore, plasma levels of the biomarker d-Dimer, a marker of hypercoagulation, showed a 2- to 3-fold increase either during painful crisis or at steady state in patients with SCD as compared to that in healthy participants, suggesting an increased risk of thrombosis.     

Increased Levels of Circulating ICAM-1, E-Selectin, and IL-2 Receptors in Celiac Disease

Adhesive interactions between endothelium and circulating cells are crucial for the development of inflammatory reactions. We found significantly higher serum levels of soluble intracellular adhesion molecule-1 (sICAM-1, 492.5 ± 22.1 ng/ml) in patients with active celiac disease (including IgA-deficient patients) than in patients on a gluten-free diet (335.7 ± 20.0 ng/ml) (P < 0.001) and healthy controls (207.4 ± 11.2 ng/ml) (P < 0.001). The concentration of soluble E-selectin in sera from celiac patients (37.2 ± 3.4 ng/ml) was also higher (P < 0.001) than in sera from healthy controls (15.5 ± 0.7 ng/ml) but, in contrast to sICAM-1, it remained high in the patients after treatment (30.2 ± 2.7 ng/ml). Interestingly, the concentration of circulating soluble interleukin-2 receptors, molecules indicating lymphocyte activation, was only increased in sera from patients with active celiac disease (2943.0 ± 214.1 pg/ml), and the level in sera from treated patients and healthy controls was comparable (1936 ± 349 and 1416 ± 111.7 pg/ml). The elevated serum level of soluble cell adhesion molecules could be used as a supplementary, noninvasive procedure for monitoring intestinal immune reactions.     

Abnormalities of coagulation and fibrinolysis in homozygous sickle cell disease

Abnormalities of coagulation and fibrinolysis were studied in a group of 28 children and young adults with homozygous sickle cell disease (SCD), either in the steady state (n = 12) or during painful crisis (n = 16). Coagulation was explored by standard clotting tests and by measurement of prothrombin complex factors, factor VIII (VIII:C) and antithrombin III (ATIII), protein C (PC) and protein S (PS) activities, while fibrinolytic potential was evaluated using D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) assays. In SCD patients, thrombin time (TT) was constantly shortened, both in the steady state (ratio to control 0.83 ± 0.08, p < 0.0001) and in crisis (0.76 ± 0.06, p < 0.0001). Mean levels of prothrombin complex were similar in asymptomatic patients to those in controls, but were significantly decreased during sickle cell crisis (p < 0.05 for factor V and p < 0.0001 for factors II,VII and X). Factor VIII:C was significantly increased, both in the steady state (207 ± 35%, p < 0.0001) and during crisis (208 ± 34 %, p < 0,0001). PS activity was reduced in the steady state (81 ± 12%, p < 0.01) and further diminished in crisis (68.5 ± 27.5%, p < 0.001), while D-dimers were significantly elevated during sickle cell crisis (1028 ± 675 ng/ml, p < 0.001). In all SCD patients, baseline levels of t-PA antigen were comparable to those in controls, whereas concentrations of PAI-1 antigen were significantly increased, either in the steady state (89.7 ± 26.3 ng/ml, p < 0.0001) or in crisis (75.0 ± 24.8 ng/ml, p < 0.0001). These results provide evidence for the presence of circulating activated clotting factors in SCD and for an imbalance of the profibrinolytic and antifibrinolytic systems most likely due to increased PAI-1 levels.     

Increased plasma levels of soluble vascular cellular adhesion molecule-1 in patients with chest pain and angiographically normal coronary arteries

AIMS: To assess plasma levels of vascular cellular adhesion molecule-1, a marker of endothelial dysfunction, in patients presenting with coronary syndromes submitted to coronary angiography. METHODS AND RESULTS: Plasma levels of soluble vascular cellular adhesion molecule-1 were measured by enzymatic immunoabsorbent assay in eight patients with angina-like chest pain and angiographically normal coronary arteries; in 14 patients with stable angina and in 18 patients with unstable angina, both with coronary lesions by angiography, and in 10 healthy volunteers. Levels of soluble vascular cellular adhesion molecule-1 were higher in unstable angina patients (1777+/-161 SE pg ml(-1)) compared to patients with stable angina (1178+/-206 SE pg ml(-1), P<0.05). Moreover, patients with angina-like chest pain and normal coronary arteries had significantly higher soluble vascular cellular adhesion molecule-1 levels (2307+/-295 SE pg ml(-1)) compared to stable angina patients (P<0.05), but similar levels compared to unstable angina patients. Patient groups had higher values of soluble vascular cellular adhesion molecule-1 compared to the control group (734+/-97 SE pg ml(-1)). CONCLUSIONS: Increased levels of soluble vascular cellular adhesion molecule-1 are associated with coronary artery disease in patients with anatomically established lesions. In patients free of flow-limiting lesions and angina-like chest pain, high levels of this marker may indicate endothelial dysfunction.     

Arterialization of Venous Blood for Differentiation of Sickle Cell Subjects in Vaso-occlusive Crisis

These studies were designed as two experiments. Experiment 1 was performed to validate the hypothesis that oxygen saturation of the venous blood may be a marker for vaso-occlusive crisis (VOC) in sickle cell patients undergoing hydroxyurea (HU) treatments. Experiment 2 was performed to test the hypothesis that an acute increase in the blood nitric oxide (NO) concentration by administering HU modulates the perception of pain in sickle cell subjects in VOC. The percent saturations of oxyhemoglobin (%O<PRE>2</PRE>Hb), reduced hemoblogin (%RHb), carboxy-hemoglobin (%COHb), met-hemoglobin (%MHb), fetal hemoglobin (HbF), and nitric oxide metabolites were measured in venous blood samples collected from sickle cell disease (SCD) who were on and off HU and O<PRE>2</PRE> at steady state and during VOC. The results showed the ratio of %O<PRE>2</PRE>Hb/RHb in VOC+HU was significantly higher than patients in the steady state who were on and off of HU (p<0.05). The %COHb was higher in all SCD groups, %COHb values were significantly different in SCD at steady state who were on HU. HU and O<PRE>2</PRE> treatment did not play important role on venous blood %O<PRE>2</PRE>Hb and pain scores in SCD during VOC. A single oral dose of HU was associated with a significant increase in the venous concentration of nitric oxide metabolites (NOx), p<0.05. These findings suggest that the ratio %O<PRE>2</PRE>Hb/RHb in venous blood and pain scores differentiate HU-untreated and HU-treated at steady state subjects from HU-treated subjects in VOC; however, the acute increase in venous NOx produced by administering HU to HU-treated subjects in VOC does not explain this difference.     

Placenta growth factor activates monocytes and correlates with sickle cell disease severity

Sickle cell disease (SCD) results in chronic hypoxia and secondarily increased erythropoietin concentrations. Leukocytosis and activated monocytes are also observed in SCD in absence of infection or vaso-occlusion (steady state), the reasons for which are unknown. We found that erythroid cells produced placenta growth factor (PlGF), an angiogenic growth factor belonging to the vascular endothelial growth factor (VEGF) family, and its expression was induced in bone marrow CD34+ progenitor cells in the presence of erythropoietin. Furthermore, the steady state circulating PlGF levels in subjects with severe SCD (at least 3 vaso-occlusive crises [VOCs] per year) were 18.5 +/- 1.2 pg/mL (n = 9) compared with 15.5 +/- 1.2 pg/mL (n = 13) in those with mild SCD (fewer than 3 VOCs per year) and 11.3 +/- 0.7 pg/mL (n = 9) in healthy controls (P <.05), suggesting a correlation between PlGF levels and SCD severity. In addition, PlGF significantly increased mRNA levels of the proinflammatory cytochemokines interleukin-1beta, interleukin-8, monocyte chemoattractant protein-1, and VEGF in peripheral blood mononuclear cells (MNCs) of healthy subjects (n = 4; P <.05). Expression of these same cytochemokines was significantly increased in MNCs from subjects with SCD at steady state (n = 14), compared with healthy controls. Of the leukocyte subfractions, PlGF stimulated monocyte chemotaxis (P <.05, n = 3). Taken together, these data show for the first time that erythroid cells intrinsically release a factor that can directly activate monocytes to increase inflammation. The baseline inflammation seen in SCD has always been attributed to sequelae secondary to the sickling phenomenon. We show that PlGF contributes to the inflammation observed in SCD and increases the incidence of vaso-occlusive events.     

Adhesion of sickle red blood cells and damage to interleukin-1 beta stimulated endothelial cells under flow in vitro

Two factors that are hypothesized to contribute to vasoocclusive crises in sickle cell anemia are increased sickle red blood cell-endothelial cell interactions and damage to endothelium. Despite considerable study, the mechanisms by which erythrocyte-endothelial interactions occur and the role of endothelial damage have not yet been fully elucidated. In this report, we demonstrate that adhesion and damage may be related in a model of vasoocclusion in sickle cell anemia. Phase contrast microscopy coupled to digital image processing was used to determine the adhesion of sickle red blood cells to 1-, 4-, and 24-hour interleukin-I beta (IL-1 beta) stimulated endothelial calls in a parallel plate flow chamber. Morphological alterations to activated endothelial cells after the perfusion of sickle erythrocytes were also identified. Pretreatment of monolayers with 50 pg/mL of IL-1 beta for 1, 4, and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. Results with an Arginine-glycine aspartic acid (RGD) peptide and monoclonal antibodies indicated a role for three different endothelial cell receptors: alpha v beta 3 after 1 hour of IL-1 beta stimulation; E- selectin after 4 hours of IL-1 beta stimulation; and vascular cell adhesion molecule-1 after prolonged exposure to cytokines. Perfusion of sickle, but not normal, erythrocytes resulted in alteration of endothelial morphology. Approximately 6% to 8% damage was observed on 4- and 24-hour IL-1 beta stimulated endothelial cells after the perfusion of sickle cells. Damage to 24-hour activated endothelial cells showed a positive correlation (r = .899) with the number of adherent sickle erythrocytes.     

Enhanced pulmonary and systemic response to endotoxin in transgenic sickle mice

Some suggest that sickle cell disease (SCD) is associated with a "proinflammatory state" that predisposes patients to acute chest syndrome in the setting of triggering factors. Conflicting data emerged when inflammation markers in SCD were compared with healthy individuals. Therefore, we examined transgenic sickle and control mice at baseline and with endotoxin (LPS) intraperitoneal injection to determine whether a proinflammatory state truly exists. At baseline, sickle mice had elevated levels of circulating leukocytes and soluble vascular cell adhesion molecule 1 (sVCAM-1). No other differences were observed at baseline or in response to saline. However, LPS challenge was associated with significant increases in mortality (p<0.05), airway tone (p<0.03), serum and bronchoalveolar lavage levels of cytokines tumor necrosis factor-alpha (p<0.03), interleukin-1beta (p<0.02), and sVCAM-1 (p<0.01) in sickle mice compared with control subjects. Furthermore, 4 hours after LPS, microarray analysis identified 413 genes differentially expressed in the sickle mice (n=5) compared with only 7 in the control subjects (n=5). No difference in lung parenchyma was observed by light microscopy. This enhanced response to LPS suggests that the sickle red blood cell confers a subclinical "proinflammatory state." This enhanced response to inflammatory insult, particularly by adhesion molecules such as sVCAM-1, could play a role in the increased susceptibility to pulmonary dysfunction that has been observed clinically in SCD.