Track topics on Twitter Track topics that are important to you
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.
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
Assistance Publique - Hôpitaux de Paris
Published on BioPortfolio: 2014-08-27T03:17:04-0400
Absorption, distribution, metabolism, and excretion (ADME) study with 14C in healthy young men.
The purpose of this study is to understand how consuming healthy cookies every day for two weeks will affect different types of fat in the blood. Specifically, the overall goal of this stu...
To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of IMA-026 administered subcutaneously (SC) or intravenously (IV) in healthy adults.
Using an operational continuum of healthy aging developed by U.S. researchers, we sought to estimate the prevalence of healthy aging among older Spaniards, inform the development of a definition of he...
Unhealthy eating behaviors contribute to obesity and chronic illness. This study examined the relative contributions of a healthy-eater self-schema (a self-conception as a healthy eater) and nutrition...
China has the largest population in the world, and its health levels have greatly affected the healthy development of the population of the world. Healthy China 2030 is a breakthrough for ensuring tha...
Recent evidence demonstrated that complex regional pain syndrome (CRPS) is associated with a larger than normal somatosensory (S1) representation of the healthy hand. The most intuitive mechanism for ...
This study assesses how the nation's preeminent health promotion and disease prevention initiative, Healthy People, is utilized by key stakeholders.
Healthy People Programs are a set of health objectives to be used by governments, communities, professional organizations, and others to help develop programs to improve health. It builds on initiatives pursued over the past two decades beginning with the 1979 Surgeon General's Report, Healthy People, Healthy People 2000: National Health Promotion and Disease Prevention Objectives, and Healthy People 2010. These established national health objectives and served as the basis for the development of state and community plans. These are administered by the Office of Disease Prevention and Health Promotion (ODPHP). Similar programs are conducted by other national governments.
Governmental guidelines and objectives pertaining to public food supply and nutrition including recommendations for healthy diet and changes in food habits to ensure healthy diet.
Surgical reinnervation of a denervated peripheral target using a healthy donor nerve and/or its proximal stump. The direct connection is usually made to a healthy postlesional distal portion of a non-functioning nerve or implanted directly into denervated muscle or insensitive skin. Nerve sprouts will grow from the transferred nerve into the denervated elements and establish contact between them and the neurons that formerly controlled another area.
Process of evaluating the health of a patient and determining if they are healthy enough for surgery.
Dietary patterns which have been found to be important in reducing disease risk.
Blood is a specialized bodily fluid that delivers necessary substances to the body's cells (in animals) – such as nutrients and oxygen – and transports waste products away from those same cells. In vertebrates, it is composed of blo...
Of all the types of Dementia, Alzheimer's disease is the most common, affecting around 465,000 people in the UK. Neurons in the brain die, becuase 'plaques' and 'tangles' (mis-folded proteins) form in the brain. People with Al...
Stress is caused by your perception of situations around you and then the reaction of your body to them. The automatic stress response to unexpected events is known as 'fight or flight'. Discovered by Walter Cannon in 1932, it is the release of h...