Circulating Endothelial Compartment During Normal and Pathological Aging

Blood will be harvested in citrated or EDTA anticoagulant and processed within one hour for Endothelial Microparticles Platelet (EMP), Circulating endothelial cells (CEC) and Circulating endothelial progenitors (EPC) determination.

Methods for endothelial marker determination:

- Endothelial Microparticles Platelet free plasma (PFP) will be prepared by a double step centrifugation of citrated blood at 1,500g for 15 min and 13,000 g for 2 min at room temperature. Then EMP will be enumerated by flow cytometry after labelling of PFP using monoclonal antibodies directed against endothelial antigens, such as CD144 (19). To investigate the proportion of EMP within the whole circulating MP, MP for platelet, leukocyte and erythrocyte origins will be determined using respectively, CD41, CD45, Glycophorin-directed antibodies, and the total number of phosphatidylserine expressing MP will be determined using AnnexinV binding.

- CEC will be enumerated using the consensual methodology based on an immunomagnetic separation assay (20). CEC will be isolated from EDTA whole blood using magnetic beads coated with antibodies directed against the CD146 antigen. Isolated cells will be identify using additional criteria such as morphology, size higher than 15µm, cell rosettes bearing more than 5 beads, and expression of endothelial markers (lectin binding).

- For EPC two complementary approaches will be used for determination:

1. Flow cytometry after direct immunolabelling of whole blood or the mononuclear cell fraction using monoclonal antibodies and viability marker 7AAD. Due to the lack of specific markers for EPC and the great heterogeneity of cells that recovered the features of EPC21 flow cytometry analysis will include the numeration of different cell populations corresponding to differentiation state of EPC: CD34+CD133+ cells and CD34+KDR+ cells. The circulating haematopoietic progenitors CD34+CD45+ will be also numbered.-

2. Clonogenic assays allowing the numeration of Colony Forming Unit after ex vivo culture of EPC including: - CFU-EC, which are EPC of myeloid subtype (also called early EPC) will be determined according to the previously described method of Hill et al. The non-adherent fraction of mononuclear cells will be plated on fibronectin coated dishes in Endocult® medium. the number of endothelial colonies will be counted after a 48h culture - High Proliferative Potential CFU, which are true angioblasts will be determined by Ingram's method (21). Mononuclear cells are plated on fibronectin-coated plates in EGM-2MV culture medium and late EPC colonies are enumerated after 10-15 days of culture. - three-dimension methyl cellulose assay in which the only added growth factor is VEGF. The CFU-EC are numbered after 14 days as usually performed for haematopoietic stem cells. Effect of hypoxia on the equilibrium between endothelial damage and repair According to Ingram, the normal vascular endothelium contains high proliferating potency EPC (22) suggesting that late EPC may also originate from the vessel wall. In addition, local hypoxia might also mobilize EPC (23).

We designed a protocol which specific aims are 1/ to test the effect of local hypoxia on the markers of endothelial damage/repair equilibrium 2/ to define whether in elderly patients, local ischemia can be used to mobilize EPC and to increase their proliferation potential compared to EPC isolated in resting conditions. Two groups of healthy volunteers aged 20-30 and 60-70 years from our cohorts will be submitted to forearm transient ischemia as described by Friedrich et al (23). Peripheral blood samples will be obtained before and after 10 min of venous occlusion performed with a cuff pressure midway between systolic and diastolic pressure (24). EPC, CEC and EMP will be enumerated according to the methods described supra and EPC will be tested for proliferating capacity at each time point. The arterial phenotype of conducting arteries will be evaluated, by the acute flow-mediated vasodilatation (FMD) and endothelium-independent vasodilatation (EIV) of the brachial artery (BA) in young and older healthy volunteers using a high-resolution echotracking system. The efficiency of venous occlusion will be evaluated by the release of t-PA by endothelial cells.