Therapeutic angiogenesis is area 3 within EVGN network and consists of 4 workpackages.

The specific objectives of workpackages 14 and 15 focussing on EPCs are:

1. To optimize EPC homing from the study of the transcriptome of endothelial cells and EPC relevant adhesion molecules and chemokines able to induce their reciprocal interaction. Overexpression of adhesive receptors in mouse EPC will be performed by the use of lentiviral vectors. EPC adhesion to endothelial cells will be tested in cell culture systems

2. To evaluate the effect of different adhesion proteins on EPC or endothelial cells. GFP or Lac Z labeled EPC will be injected in mice null for different adhesion proteins (such as JAM-A, VE-cadherin, ICAM-1, PECAM and double null JAM-A/PECAM).

3. To define the transcription factors involved in endothelial differentiation (embryonic versus adult), including Forkhead transcription factors (particularly Foxo1), ets-1 related transcription factors and members of the Sox family. EVGN laboratories (parners 3, 7, 21) implemented the embryonic stem cell differentiation assays.

4. To characterise the ß-catenin signalling pathway in the bone marrow stem cell niche, and identify selective mobilizers of EPC by studying the effects of Wnt antagonists, estrogen and FGF isoforms on EPC mobilisation.

5. To define the role of different proteases in vasculogenesis and angiogenesis.

6. To anlayse the functional activities of stem/progenitor cells by establishing assays to discriminate between stem/progenitor cell capacities, including integration and differentiation to endothelial and skeletal or cardiac myocytes, release of protective factors acting in a paracrine manner.

7. To compare different stem/progenitor cell populations for improvement of atherosclerosis, neovascularization and tissue regeneration. Different human cell types (endothelial progenitor cells, smooth muscle cell progenitor, peripheral blood mononuclear cells, mesenchymal stem cells, purified hematopoietic progenitor cells, adipose tissue progenitor cells) will be tested.

8. To assess the efficiency of various cytokines (G-CSF, IGF, Erythropoietin or IL-18 in comparison with VEGF or GM-CSF) to induce stem/progenitor cell mobilisation and homing.

The workpackage WP16 is aimed at developing animal models for evaluation of the efficacy of non-cell-based therapy to improve neovascularisation in ischaemic areas and to promote post-ischaemic tissue regeneration. The specific objectives are:

9. To study the effects of therapeutic injection of drugs or gene transfection in mouse experimental models of human disease (diabetes, hypertension, atherosclerosis).

10. To evaluate the interaction between the Wnt/frizzled system and VEGF in vessel formation and maintenance using VEGF on/off x sFRP-1-/- mice.

11. To evaluate the vascular consequences of myocardial infarction in the transgenic mice with conditional modulations of VEGF expression and signaling in the myocardium by induction of VEGF or of a soluble VEGF-R1 (“VEGF trap”) by tetracycline withdrawal and its shut-off by tetracycline addition, developed by partner 21.

The workpackages WP15 and WP17 are clinicaly-oriented toward the clinical evaluation of the angiogenic potential of circulating EPCs. Their objectives are:

12. To characterise bone marrow stem cell number and function in patients in order to evaluate whether risk factors influence bone marrow stem cells before mobilization. Bone marrow cells will be obtained by diagnostic bone marrow aspiration, and different stem cell subpopulation will be determined by FACS analysis. Functional activity will be assessed by colony forming assays, migration assays and invasion assays (stimulants: VEGF and SDF-1).

13. To determine the genetic profiles of EPCs from healthy controls and patients with CAD by using Affymetrix chips analysis.

14. To establish study protocol and logistics for multi-center European Cell Therapy Outcome Trial.

15. To analyse results from the REPAIR-AMI trial, including sub-studies and 1-year follow-up.

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