Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Rohban, R.
Early Signaling Signature during Stem Cell-Associated Neo-Vasculogenesis Using Proteomic Profiling
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medical University of Graz; 2014. pp. 221
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- Autor*innen der Med Uni Graz:
- Rohban Rokhsareh
- Betreuer*innen:
- Becker Jürgen Christian
- Heinemann Akos
- Altmetrics:
- Abstract:
- De novo vessel formation (neo-vasculogenesis) can be induced by co-transplantation of pericytes or their mesenchymal stem/progenitor cells (MSPC) with endothelial cells or endothelial colony-forming progenitor cells (ECFC). The precise mechanisms regulating neo-vasculogenesis in vivo are still a matter of debate which hampers the development of strategies for therapeutic intervention referred to as ‘regenerative medicine’. Here we employed a novel strategy for unraveling the molecular determinants of neo-vasculogenesis by proteomic profiling of the early signaling signature in vivo. We applied MSPC and ECFC for subcutaneous transplantation in matrix composites either alone or mixed at a ratio of 1:5 into immune deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; NSG mice. Implants were harvested 24h after transplantation for proteomic profiling and the protein content of the transplants was subjected to KAM 1.3 antibody microarray (www.kinexus.ca) testing over 800 signaling and phospho-proteins. The state of vessel formation and stability of the transplants were verified in parallel by histological follow-up of corresponding explants harvested 2, 8 and 24 weeks after transplantation. Significantly expressed targets were pre-selected from antibody microarray data based on differential display and were used for in vitro toxicity and viability assays as well as in vivo modulation of induced neo-vasculogenesis. Results confirmed that co-transplantation of ECFC with MSPC was most efficient at forming stable-perfused human vessels. “ECFC only” explants showed vascular structure formation only after transplantation of higher cell numbers and later in the time course after transplantation. Early chondrogenesis state was detected in ”MSPC only“ explants after 8 weeks. Analysis of protein microarray data revealed significant alteration of components including (1) caspases, death protein-6 (DAXX) and p53 usually involved in death-associated signaling pathways, (2) epidermal growth factor receptor 2 (Her2/ERB), mitogen activated protein kinase (MAPK), mammalian target of rapamycin (mTOR) and transforming growth factor- beta (TGF-ß), (3) focal adhesion protein kinase (FAK), vascular endothelial growth factor (VEGF), JAK-STAT and Wnt. Involvement of selected candidates from the caspase cascade in stem/progenitor cell-mediated neo-vasculogenesis was further assessed using in vitro and in vivo caspase blocking strategies which led to diminished capillary-like network structure formation in vitro as well as significant decrease in vessel formation in vivo. Co-transplantation of ECFC and MSPC with the defined 5:1 ratio or sole ECFC with a higher cell dosage was essential for vessel generation in vivo. Proteomic profiling unraveled independent and partially overlapping signaling networks involved in the complex process of vascular regeneration. Pharmacological blockade of caspases as significantly up regulated proteins and caspase-4 as one selected target in the proteomic profile of early vasculogenesis was shown to hamper endothelial network formation in vitro and vasculogenesis in vivo. Proteomic profiling unraveled a signaling signature which can be targeted to modulate neo-vasculogenesis in vivo and may lead to more efficient tissue engineering methods.