Investigating the Roles of Macrophages in Vessel Development Utilizing Poly(Ethylene Glycol) Hydrogels

Macrophages, key cells of the immune system, are often present at active sites of angiogenesis. It has been found that macrophages can play a critical role in supporting blood vessel development as the removal of these cells results in impaired vessel development. Because of the supportive role macrophages can play in vessel formation, macrophages can be considered as a novel cell source to support vessel development

Within the field of tissue engineering, one major limitation towards the development of large scale tissues is the need for vascularization of the tissues to support oxygen and nutrient demands. The need for vascularization combined with the roles of macrophages in vessel development introduce a unique opportunity to utilize cells of the immune system (in this case, macrophages) to support vessel development within tissue engineered constructs. In this thesis, we identify the roles of macrophages in vessel development utilizing a cell-adhesive and proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogel.

In our initial studies, we introduced the notion that macrophages enhance vessel formation of endothelial cells when both cells are simultaneously encapsulated into the PEG-based hydrogel. We next assessed the macrophage response to the presence of endothelial cells in our PEG-based hydrogel. Macrophages became more spread depending on the density of endothelial cells they were encapsulated with. We found that a 1:1 ratio of endothelial cells to macrophages resulted in the most spread population of macrophages within the PEG-based hydrogels. Macrophages also closely associated with endothelial cells in a proximity dependent manner; macrophages closest to endothelial cells were more spread than macrophages further away from the endothelial cells. We next classified the types of associations seen between the macrophages and the endothelial cells: macrophages closely associating with endothelial cells and macrophages bridging neighboring endothelial cells. The close association seen between the macrophages and endothelial cells mimics the close contact seen between endothelial cells and support cells. The bridging association seen mimics the cell-chaperoning behavior of macrophages during in vivo vessel formation. It has been seen that macrophages can physically connect two endothelial tip cells, thus acting as the cell-chaperone. The bridging association seen in this work complements the cell-chaperone behavior seen in vivo.

This work also explored the roles of macrophage phenotypes in governing the role of macrophages in vessel formation. Macrophages are highly plastic cells that alter their function based on environmental cues. There are two main paradigms of macrophage phenotypes: M1, pro-inflammatory macrophages, and M2, pro-tissue healing macrophages. This work explored the roles of macrophage phenotypes to vessel formation in the PEG-based hydrogels. M0 and M2 macrophages were found to support vessel development when encapsulated with endothelial cells. M1 macrophages significantly retarded vessel formation when encapsulated with endothelial cells. The endothelial cell and M2 macrophage co-culture secreted VEGF while the M1 macrophages retarded endothelial cell proliferation.

Due to the diverging effects of macrophage phenotype on vessel formation, we developed of PEG-based hydrogels capable of presenting a stimulating microenvironment to macrophages and endothelial cells. We found that a M2 stimulating hydrogel enhanced vessel formation when endothelial cells and macrophages were encapsulated in the hydrogel.

Overall, this dissertation demonstrates the role of macrophages in supporting vessel formation in PEG-based hydrogels. Findings in this thesis have helped to elucidate the diverging roles of macrophage phenotypes in supporting vascularization of PEG-based hydrogels. Moreover, this work has created PEG-based materials can be manipulated macrophage phenotype. This work highlights the usefulness of macrophages in vessel development and the usefulness of a macrophage-directing platform to enhance vascularization of tissue engineered constructs.