Circulating Endothelial Compartment During Normal and Pathological Aging
Condition: Healthy
Study type: Observational

Summary: 
      This part of the project aims to describe changes in markers of vascular competence
      (Endothelial Microparticles Platelet (EMP), Circulating endothelial cells (CEC) and
      Circulating endothelial progenitors (EPC)) at different ages of lifespan both in
      physiological state and in response to hypoxia-induced vascular stress.
    

Detailed description: 
      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.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        healthy volunteers
      
Biospecimen description: 
      whole blood
    
Keywords: Circulating endothelial compartment; microparticles; endothelial progenitors cells; circulating endothelial cells; transcriptome; aging; Healthy volunteers