UBC Theses and Dissertations
Characterization and use of a controlled in vivo model for angiogenesis Heine, Heather Lynn
In this thesis, I attempted to better understand progenitor cells participating in neoangiogenesis. To acheive clarity regarding the identity of endothelial progenitor cells (EPCs) that may contribute to new blood vessels, I used multicolor flow cytometry to compare cells positive for commonly cited EPC surface proteins. Cells positive for 2-3 markers could not be reliably isolated due to limitations of techniques available. Furthermore, the absence of a convincing functional assay to discriminate cell types made the significance of positive results unclear. This initial experience led to studies of a murine subcutaneous model of angiogenesis employing a custom-built chamber. I combined this tool with the use of an automated high-resolution imaging system to establish baseline parameters of vessel growth in vivo. Chamber parameters were established to create a baseline for cell types that infiltrate during angiogenesis, rates of infiltration of each cell type, and for high-resolution morphology of newly forming vessels. In establishing the model system, I tested the hypothesis that certain manipulations markedly alter the growth of neovessels and their constituent cell populations. 1) Purified EPC populations were implanted within the chamber to test their potential to participate in the neoangiogenesis. After an apparently successful pilot project of implanting Sca-1+ cells within chambers, despite six successive studies, the activity of purified cells involved in neoangiogenesis was not confirmed. Alternate approaches to introduce sorted subsets of cells are suggested to continue these studies. 2) Bone marrow (BM) transplants with green fluorescent protein (GFP)-labeled cells were undertaken to examine BM-derived cell contributions to neoangiogenesis. The majority of BM-derived cells within the chamber were F4/80+ white blood cells. In rare instances GFP+CD31+ cells were evident. 3) CD34 is an important sialomucin widely used for EPC isolation however its function remains unclear. Chambers implanted within CD34 null mice show differences in the rates of penetration and cell density in Hoechst 33342+, CD31+ and NG2+ cells, and endothelial tubule formation appears to be defective. The characterization of the model system that I achieved now provides a stepping-stone for studies of specific cells, growth factors, and environmental components best suited to support therapeutic efforts in neoangiogenesis.
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