The engraftment problem: TNFalpha increases adhesion and migration of endothelial progenitor cells to vascular endothelial cells

The purpose of this thesis is to help elucidate mechanisms by which EPCs engraft into the vasculature. More specifically, studies have been designed that test the mechanistic role of TNFalpha (a proinflammatory cytokine) in mediating both migration of EPCs towards vessels, and binding of EPCs to the inner most layer of the vasculature, the endothelium. Aim 1 tests in vitro the hypothesis that EPC TNFalpha treatment can increase adhesion to vascular endothelial cells (ECs) by increasing the affinity of existing bonds. To do this, a parallel plate flow chamber was constructed and used to quantify the force required to pull apart adherent EPCs/ECs. A mathematical model was developed to simulate cellular adhesion as a function of bond expression and affinity. Simulations and experimental data demonstrated that TNFalpha can increase the adhesion of EPCs to ECs through an increase in bond affinity alone.;Aims 2 and 3 focus on the mechanism by which EPCs migrate toward vessels and the mechanism by which TNFalpha mediates this process. Aim 2 tests the hypothesis that TNFalpha increases the migration of EPCs to vessel-like structures in vitro. To do this, a novel migratory assay and computational method were developed to track the locations of EPCs with respect to vessel-like structures in vitro. It was demonstrated that EPC TNFalpha treatment increased the migration of EPCs towards vessel-like structures. Aim 3 proposes and tests a novel molecular mechanism of TNFalpha mediated migration in EPCs. To study this, a combination of genomic and proteomic studies were utilized to identify potential candidate mechanisms of action. The candidates were ranked and TNFalpha-induced upregulation of CADM1 was hypothesized as the mechanism responsible for increased migration. To test this hypothesis, expression of CADM1 was inhibited using siRNA and the involvement of CADM1 in the migratory process was tested by repeating the migratory assay from Aim 2. In the cells where CADM1 expression was inhibited, TNFalpha-mediated migration was attenuated, confirming the role of CADM1 in the migratory process.;The results of this thesis are important when designing trials of cellular based therapies as the majority of patients enrolled in cell based therapy clinical trials have elevated levels of TNFalpha as a component of the underlying disease pathology, so mechanisms of engraftment in response to TNFalpha are crucial to understand. Better understanding of the mechanisms of engraftment will allow clinical identification of isolated cells that have an impaired therapeutic potency. Ex vivo tests of an isolated population of cells will likely be able to predict the likelihood of successful therapy. These results will lead to better selection of potential candidates for cellular based therapy and possible stem cell treatments to enhance the function of patients' cells that are therapeutically impaired. (Abstract shortened by UMI.).