Fellowship for Randall Smith: The Role of Monocytes in the development of tissue engineered vascular grafts

Project Summary/Abstract Circulating monocytes within the blood have an essential role in the biological response to wounds and inflammation. These cells are well known to differentiate to macrophages during such processes and generally serve two roles, either the cells polarize to a pro-inflammatory phenotype and devour the damaged cells (M1 phenotype) or an anti-inflammatory phenotype (M2) and assist in regeneration of the tissue through various methods such as producing soluble cytokines and growth factors that induce nearby cells to proliferate and thus regenerate the damaged tissue. Typically these two phenotypes are sequential, in that an M1 phase dominates to clear away damaged tissue followed by a switch to the M2 phenotype to regenerate the tissue with healthy cells. Recently our group has shown the presence of monocytes on a-cellular grafts immobilized with vascular endothelial growth factor (VEGF) and heparin. Interestingly, the lumen of these blood vessels, when subjected to immunohistochemistry, revealed co-staining of the classical monocyte marker CD14 and M2 macrophage/monocyte marker, CD163, with endothelial markers CD144 and eNOS. In addition, our group has demonstrated the use of immobilized VEGF to capture endothelial and monocyte cells under a range of shear strengths, from low to physiological (0.5-15 dyne/cm2). This evidence points to the potential of monocytes to differentiate into specific target cells that may repopulate damaged tissue directly. Building on these observations, the objectives of this proposal are to evaluate the potential of circulating monocytes to endothelialize vascular grafts. It is hypothesized that VEGF is capturing monocytes from the blood flow and further inducing a trans-differentiation towards an endothelial phenotype capable of preventing thrombosis or that captured monocytes recruit rare endothelial progenitors from the blood to regenerate the endothelium. The specific aims of this hypothesis have been designed to directly test these hypotheses. Studies proposed in Aim 1 will evaluate the ability of VEGF functionalized surfaces to direct the differentiation of monocytes in-vitro towards an endothelial phenotype under static (Aim 1a) and shear (Aim 1b). In Aim 2, tissue engineered vessels will be seeded with monocytes or monocyte derived endothelial like cells from male sheep and implanted into female ovine animal models to assess the role of monocytes in maintaining patency and generation of a functional endothelium. Overall, the proposal is structured to both implement a new method for endothelializing vascular grafts and to understand the in-vivo mechanism of endothelializing A-cellular TEVs. The results from these studies will identify a new source of cells and methodology to use for directing tissue regeneration within the body.