Sickle Cell Disease and Endothelial Progenitor Cells (EPCs)

In the bone marrow, a reservoir of EPCs does exist, which can be mobilized into circulation when needed, for example during ischemia, a situation which may occur in sickle cells disease. These cells are able to reach distant targets and to participate to the neovascularisation processes necessary for tissue and vascular healing. Conversely, abnormalities of the maturation, mobilization or homing processes would contribute to the thrombotic and ischemic risks associated to ischemia. Furthermore, vascular stress such that encountered in SCD leads to the detachment of endothelial cells from the vessels. The number of detached mature circulating endothelial cells (CEC) seems to be related to vascular hurting. The balance between EPCs and CECs would thus be informative of the vascular condition in patients during the progression of the disease. A relatively high level of EPCs would indicate a prominence of a healing neoangiogenesis or vasculogenesis process, while increasing levels of CECs would be indicative severe progression of the disease. Our studies and those published by others indicate that the number of EPCs in peripheral blood is tightly regulated. Indeed, mediators of inflammation could participate to EPC mobilization and to their recruitment to the diseased areas by local activation of the vascular endothelium. In this context, sickle cells disease provokes transient occlusion episodes, leading to a situation of chronic ischemia in different organs, sometimes with acute episodes. This suggests defect of the maintenance of the vascular integrity in these target organs.Within this project, we will compare the number of EPCs and CECs in 25 SCD patients and 25 normal individuals in search of a correlation with the severity of the disease. SCD patients with potential confounding factors that may alter endothelial physiology (drugs, blood transfusions, hydroxyurea therapy) are excluded. In addition, we will analyse and compare in culture and in vivo the phenotypic and functional characteristics of EPCs obtained from patients and controls. We have a working bilateral hypothesis, since we do not know whether SCD subjects have appropriate angiogenic balance (high EPCs/CEC ratio with functional EPCs) to face the situation of endothelial activation and tissue hypoxia induced by SCD. Therefore, we will quantitatively and qualitatively study several distinct biological steps:1. Evaluate the number of colonies generated by the EPCs in culture and their kinetics of appearance in patients (major end point)2. Enumerate EPCs and CECs by flow cytometry using a specific set of markers (CD133, CD146, KDR).3. Measure of the plasma concentrations of PlGF, VEGF, EPO, ET-1, modulators of angiogenesis.4. Test of correlations between these respective characteristics of EPCs with clinical vascular abnormalities of the disease (retinopathy assessed by angiography, nephropathy, leg ulcers) and severity of vaso-occlusive crisis assessed by number and length of hospitalizations during the previous year and during the study.

Inclusion criteria

• Age between 18 and 45 yo
• SS genotype if patient
• Absence of relative with sickle cell anemia if healthy volunteer.
• Affiliated to the national health insurance benefit
• Signature of Informed consent form
• oral contraceptive regiment with micro or mini estro-progestative (women)

Exclusion criteria

• present or past (or interrupted for less than 3 months) therapy with hydroxyurea
• Present or recent blood transfusion and/or exchange (in the past 3 months)
• SC genotype (patients)- BMI > 30 kg/m2
• Positive serodiagnosis for HIV, HVB, HVC
• Know allergy to fluorescein (patients)
• Hemoglobin < 7.5 g/dL
• Pregnancy confirmed by Beta-HCG test in plasma
• Menopause
• Extreme difficulty for adequate venous puncture
• Smoking for the last month.
• ALAT or ASAT > 2 x N
• Hypercholesterolemia (cholesterol total > 2.,2g/L or 6.55 mmol/L),
• Treatment by statin
• Diabetes (fasting blood glucose level equal or superior to 6,1 mmol/L)
• Hypertension (systolic blood pressure > 140 mmHg or diastolic blood pressure > 90 mmHg).