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SEVENTH FRAMEWORK PROGRAMME

The Project - WP 14 Vascular research, ex vivo

Objectives

1. To evaluate the number, subpopupations and angiogenic properties of circulating progenitor cells (CPCs) in patients with heart failure and/or the type 2 diabetes mellitus.

For that we plan to compare the number and subpopupations of CPCs in patients with HF and combination of HF/DM type2 and healthy volunteers, and to estimate the relationship between CPCs and the severity of HF(NYHA classes), heart function parameters, and clinical outcome during one year follow-up (Task 1); to evaluate the endothelial dysfunction in relation to the number of CPCs and CPC subpopulations in patients with HF with or without DM (Task 2); to estimate the relationship between CPCs and circulating angiogenic and antiangiogenic, proinflammatory cytokines in patients with HF and DM (Task 3); to assess of the angiogenic potential of EPCs in patients with heart failure and diabetes mellitus (Task 4).

2. To determine the functional characteristics and the angiogenic potential of adipose tissue derived stromal cells in patients with HF and DM.

We aim assessing the phenotypic characteristics, expression and secretion profiles of ASCs isolated from different sources in patients with HF and/or DM (Task 5); to evaluate the angiogenic potential of adipose stromal cells isolated from patients with DM and /or HF and molecular/ cellular mechanisms of ASCs angiogenic action(Task 6).

Results achieved within WP14 will be cross-fed to WP12 (Molecular pathways, cell culture), WP13 (Molecular pathways, cell cultures) WP 09 (Cellular research on human fat). Data cumulated at executing Tasks 1-6, WP14 (together with data provided by WP13, (Cellular research on human fat) and WP12 (Molecular pathways, cell culture)) will provide an essential foundation for successful execution of Task 3 of WP13 (creation of a system of criteria allowing estimating a regenerative potential of the own stem cells of the heart failure and/or diabetes mellitus patients) and Task 4 of WP13 (to design and execute clinical trials aiming assessing the established system of criteria), which is critical for ultimate success of the overall project. Potential impact through the development, dissemination and use of the results obtained under execution of WP14 will be realised in developing new cell therapy approaches for HF based on using own ASCs as well as CPCs.

Workpackage Leader: Moscow CRC
Involved Partners: St. Petersburg, Moscow MSU, Tomsk

Workpackage description

Objectives 1. To evaluate the number, subpopupations and angiogenic potential of circulating progenitor cells (CPCs) in patients with heart failure and/or the type 2 diabetes mellitus.

Despite improved outcome bless to the development of new pharmacological and mechanical treatment modalities, HF continues to be a prevalent disease with substantial morbidity and mortality. The important clinical markers of an adverse outcome in HF are related comorbidities including renal disease, anaemia and diabetes mellitus. HF is characterized by peripheral and myocardial tissue ischaemia and endothelial dysfunction that is correlated with disease severity. This in turn results in systemic release of angiogenic factors and activation of multiple neurohormonal axes that may promote the peripheral mobilisation of endothelial progenitor cells EPCs from the bone marrow niche in an attempt to facilitate a process of angiogenesis and myocardial regeneration. Cumulative evidences suggest that patients with diabetes may suffer from decreased amounts and impaired function of peripheral circulating EPC. The metabolic alterations including oxidative stress, hyperglycemia despite normal or high insulin levels, inflammation, disbalance in growth factors and cytokines levels observed in HF and DM may act independently in concert to impair progenitor cells functionality. The overall hypothesis of this objective is that poor prognosis in patients with combination of HF and DM has been attributed although partly to impaired function of circulating progenitor cells responsible for heart neovascularisation and regeneration. Assessment of circulating progenitor cell properties in patient with HF is important for estimating their therapeutic potential and prediction of an effectiveness of the own stem cell therapy.

Task 1. (months 13 - 36) Compare the number and subpopupations of CPCs in patients with HF (ischemic cardiomyopathy) and combination of HF/DM type2 and healthy volunteers.

Work will be performed in a selected group of patients with ischemic cardiomyopathy and HF of different NYHA classes with or without DM. As control group healthy volunteers of the same age and sex will be studied.

The presence of the different CPCs subpopulation including total CD34+ cells, endothelial progenitor cells (EPC) assessed both as CD34+ CD45- cells coexpressing AC133 and vascular endothelial growth factor (VEGF) receptor-2 and the early tissue-committed cells expressing cardiac, muscle, and endothelial markers (CD34+, CD117+, CXCR4+, c-met+, CD34/CD117+, and CD34/CXCR4+ cells) in populations of mononuclear cells in peripheral blood will be assessed using flow-cytometric techniques. The relation of total CPCs and subpopupations number to the NYHA classes, B-type natriuretic peptide (ELISA ) and parameters of heart function and clinical outcome during one year follow-up will be evaluated to clear up whether CPCs could be marker of HF severity. This task will be fulfilled in MCRC (patient characterization and collection, blood collection) and MSU (flow-cytometric analisys).

Task 2. (months 13 - 36) To evaluate the endothelial dysfunction in relation to the number of CPCs and CPCs subpopulations in patients with HF with or without DM.

Parameters of endothelial dysfunction such as brachial artery flow-mediated vasodilatation, carotid IMT, distensibility coefficient, stiffness parameters, selectins levels, endotelin-1level (ELISA), NO metabolites serum levels (HPLC) will be evaluated in patients with HF and DM in relation CPC numbers as well as to the NYHA classes and parameters of heart function. This task will be fulfilled in CRC.

Task 3. (months 13 - 36) To estimate the relationship between CPCs and circulating angiogenic and antiangiogenic, proinflammatory cytokines in patients with HF and DM.

Plasma levels of TNF-alpha, VEGF, HGF, TGF-beta, stromal derived factor-1 (SDF-1), granulocyte-colony stimulating factor (G-CSF), adiponectin and leptin (ELISA) will be assessed in patients with heart failure and the type 2 diabetes mellitus in relation to CPCs number, the NYHA classes, B-type natriuretic peptide (ELISA ) and parameters of heart function.

All the parameters studied in tasks 1-3 will be correlated with HF severity and clinical outcome. This task will be fulfilled in CRC.

Task 4. (months 13 - 36) Evaluation of the angiogenic potential of EPCs in patients with HF and DM.

Work will be performed on the selected groups of patients and control subjects. EPCs will be isolated from peripheral blood using preparative cell sorting or immunomagnetic purification and will be cultured in appropriate conditions. EPC ability to express angiogenic factors and their receptors will be compared in different patients groups.

Task 4 will be fulfilled by MSU. CRC and MSU will interact closely in working over objective 1, with available technical platforms and personnel expertise utilized to achieve rapid progress of the study. These data will also be utilized in WP13 for understanding possibilities of using own progenitor cells for cell therapy of patients with HF and DM.

Objectives 2. To determine the functional characteristics and the angiogenic potential of adipose stromal cells in patients with HF and DM.

The description of pluripotent cells in adipose tissue has led to the concept that adipose tissue may provide a novel autologous source of cells with significant potential for tissue regeneration. A growing body of literature suggests that human ASCs possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. Indeed, it was demonstrated that these pluripotent cells which reside in the "stromal" or "non-adipocyte" fraction of the adipose tissue have the capacity to differentiate in culture into adipocytes, chondrocytes, osteoblasts, neuronal cells, endothelial cells, myotubes and cardiomyocytes. It was also shown that ASCs stimulate angiogenesis and improved tissue perfusion in a model of hind limb ischemia suggesting that ASCs may be a potential tools for therapeutic angiogenesis. We have shown previously that ASCs secrete significant amounts of angiogenic growth factors like VEGF, HGF, bFGF as well as pro-inflammatory cytokines. Their secretory activity profile considerably changes in hypoxia condition. One can suggest that the secretory products of ASCs as well as the adipocytes can influence heart function. Moreover there are some evidences that human epicardial adipose tissue might contribute locally to the pathogenesis of coronary atherosclerosis by producing proinflammatory cytokines and adipokines. To develop HF cell therapy strategy based on using own ASCs we need to evaluate the angiogenic and regenerative potential of ASCs in patients with HF with and without DM.

Task 5. (months 13-46)

Studies of the phenotypic characteristics, expression and secretion profiles of ASCs isolated from different sources in patients with HF and/or DM will be performed on this stage of WP14. ASCs will be isolate from subcutaneous adipose tissue and pericardial adipose tissue of patients with HF and/or DM obtained during by-pass surgery. The immunophenotypic characteristics (the proportion of CD34+CD31-, c-kit+ cells etc.), will be assessed using flow-cytometric techniques and immunohistochemistry. The expression profile and secretion of GFs, cytokines including pro-inflammatory cytokines and adipokines by these cells will be assessed by using RNA and protein super-arrays as well as real-time PCR and ELISA. For the selected group of patients with postinfarction LV aneurism and HF who underwent cardiac surgery for the resection of aneurism the expression profile and content of GFs, pro-inflammatory cytokines and adipokines, progenitor cells (CD34+, c-kit +) as well as macrophages, lymphocytes, and basophils tissue distribution will be studied in a total epicardial adipose tissue using RNA and protein super-arrays, immunohistochemistry with fluorescence and confocal microscopy. This task will be fulfilled in CRC and MSU.

Task 6. (months 13-54)

At this stage of WP14 we plan to study the effect of ASCs on the formation and stabilization of vessel-like structures by endothelial cells and EPC using co-culture system in vitro and Matrigel implant model in vivo. Rationale: Utilizing Matrigel implant model we have showed recently that hypoxia-treated ASCs stimulated angiogenesis as well as vessel maturation of the newly formed blood vessels in vivo. We and others also demonstrate that ASC contain a population of NG2-positive cells, corresponding to pericytes. To elucidate mechanisms of ASCs angiogenic action we suggest using a co-culture model of ASCs cells and endothelial cells/endothelial progenitor cells. Human and rat cells will be used in these experiments. The formation, stability and branching of CD31- positive vessel-like structures in vitro will be assessed in a co-culture model of ASCs cells and endothelial progenitor cells. The role of paracrine activity of ASC cells will be assessed by comparing the effect of ASCs condition medium with the effect of ASCs on vessel structures formation. The role of cell-cell contacts as well as participation of ASCs in the formation of vessel structures in co-culture with EPCs will be assessed using immunofluorescent analysis and confocal microscopy, and also by labeling ASCs with lipophylic fluorescent dye CM-DiI-Cell Tracker. These experiments will test the possibility to stimulate the angiogenic capacity of EPC by ASCs and will clear up the mechanisms of ASCs action including secretion of factors stimulating angiogenesis and vessel maturation as well as formation of cell-cell contacts which could be beneficial for the stimulation of vascularization in ischemic tissues by ASCs administration. Using Matrigel Implants model in immunodeficient mice we will test the capacity of ASCs isolated from patient with HF and /or DM to stimulate the vascularisation of the Matrigel. We also will test whether ASCs from healthy donors could improve angiogenic properties of EPC from patient with DM and/or HF. This task will be fulfilled in CRC and MSU.

Experience and data cumulated at executing Tasks 6, will provide an additional foundation for Task 2 (WP13) focused on studying the "reversibility" of cellular models of heart failure, diabetes mellitus, obesity and cachexia.

WP 14 Vascular research, ex vivo