Sickle Cell Disease and Endothelial Progenitor Cells (EPCs)
Sickle Cell Disease and Endothelial Progenitor Cells (EPCs)
Sickle Cell Disease (SCD) is the most prevalent genetic disease of haemoglobin.The underlying
 abnormality in the red blood cell (RBC) of SCD is the presence of abnormal sickle cell
 hemoglobin (HbS), which, when deoxygenated, becomes relatively insoluble, forms aggregates
 with other hemoglobin molecules within the RBC and causes rigid deformation of the cell.
 Acute pain vaso-occlusive crisis, strokes and acute chest syndrome are the main acute
 complications, sometimes life-threatening, often leading to organic and functional squeal.
 Although the common SS form of SCD is a unique gene disorder, the range of the clinical
 severity is remarkably wide and striking, suggesting that clinical polymorphism is due to
 modifier genes and environmental factors.Most of the research efforts have been focused on
 the biology of haemoglobin and of the red cells. Meanwhile, the complex pathophysiology of
 SCD is undoubtedly influenced by the many physiologic functions of the vascular wall. In line
 with this hypothesis are several reports of increased circulating levels of
 endothelium-derived surface molecules in SCD patients suggesting marked endothelial stress in
 SCD. Similarly, other processes that involve the endothelium, such as leukocyte adhesion and
 activation, may play a role in vascular occlusion. This accumulation of data raises the
 unanswered question of the mechanisms of endothelium maintenance and regeneration in SCD.
 Through these mechanisms, it is likely that function or dysfunction of the vascular
 endothelium contributes to the overall vascular pathobiology of this disease, which includes
 recurrent vaso-occlusions, stroke, leg ulcers, chronic organ ischemic damages, and
 neovascularizing retinopathy that affect nearly one-half (48%) of the surviving patients by
 the fifth decade.Thus, our groups have combined their respective clinical and biological
 expertises to test the hypothesis that SCD is a condition of specific endothelial stress and
 dysfunction upon chronic and Paracystic abnormal interactions with circulating cells and
 abnormal oxygen delivery to tissues. Specifically, we hypothesize that chronic endothelial
 stress with detachment of activated endothelial cells require increases mobilisation of the
 Endothelial Progenitor Cells (EPCs) that maintain endothelial homeostasis to avoid major
 thromboembolic events and vasospasm. Inappropriate mobilisation or maturation of the EPCs in
 SCD may participate to the severity of the disease.
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).