BACKGROUND: Loss of the trachea and proximal airway from congenital disorders, acute injury or chronic disease is a life-threatening complication. The lack of suitable native replacements has led to attempts to bioengineer tracheas. Recent clinical cases of bioengineered tracheal transplants have been hampered by an inability to maintain growing cell populations. The majority of cases have ended in significant acute and chronic complications, morbidity and death. A growing body of literature has found that a ligand-receptor interaction between the secreted protein thrombospodin-1 (TSP1) and its high affinity receptor CD47, which is upregulated in tissue injury, constitutively restricts multiple transcription factors that modulate self-renewal and pluripotency. ududHYPOTHESIS: TSP1-CD47 signaling is maladaptively induced in injured airways and bioengineered tracheas to inhibit cellular dedifferentiation, proliferation and restoration of engineered tracheal transplants. ududMETHODS: This dissertation first describes a murine orthotopic tracheal transplant model and second describes methods to characterize the effects of TSP1-CD47 signaling on airway epithelial cells. In vitro, airway epithelial cell wound healing and tissue angiogenic capacity was assessed using a variety of techniques. In vivo, naphthalene injury and orthotopic transplantation of decellularized tracheal grafts was performed in wild-type mice or in mice that lacked TSP1-CD47 signaling, and epithelial restoration was assessed. ududRESULTS: Decellularized tracheal grafts support re-epithelization within seven days and epithelial differentiation within eight weeks after transplantation. Disruption of TSP1-CD47 signaling restores self-renewal gene expression in tracheal epithelial cells exposed to hypoxic stress and stimulates more rapid wound healing in an in vitro model of airway injury. TSP1 inhibits proliferation of airway epithelial cells while antibody blockade of CD47 restores cell proliferation. Further, TSP1 is upregulated following airway epithelial cell seeding onto decellularized tracheal scaffolds. TSP1-CD47 signaling is induced in the airways of WT mice after injury. CD47 null mice displayed enhanced tracheal healing of both chemically injured airways and following orthotopic transplantation of decellularized tracheal grafts. ududCONCLUSION: TSP1-CD47 signaling is maladaptively upregulated in stressed tracheal epithelial cells, in injured airways, in cells seeded on decellularized tracheal grafts, and in engineered tracheal transplants to limit self-renewal gene expression and inhibit cell proliferation, airway healing and cellular incorporation of bioengineered tracheal transplants.
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