Negative Regulators of Angiogenesis during Wound Healing

During dermal wound repair, hypoxia-driven proliferation results in dense but poorly-perfused, disorganized microvascular networks, similar to those in tumors. Concurrently, activated dermal fibroblasts generate an angio-permissive, provisional extra-cellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for re-establishing tissue homeostasis. However, the mechanisms guiding wound resolution are poorly understood. In this Thesis, we explored the roles of two candidate endogenous anti-angiogenic factors, Sprouty-2 and Pigment Epithelium-Derived Factor (PEDF), in the regulation of wound resolution. Spatial and temporal expression patterns of the two factors were characterized in a reproducible model of excisional wound healing in the mouse. Sprouty-2 was found to be an intra-cellular protein associated with cells in the wound bed and its expression peaked during the proliferative phase of healing. PEDF was found to be a fibroblast-produced matricellular protein that localized with microvasculature and its expression peaked during the remodeling phase of healing. In a series of controlled in vivo experiments, wherein levels of Sprouty-2 or PEDF in wounds were altered via exogenous addition of recombinant proteins or antibody inhibition of endogenous proteins, the anti-angiogenic roles of candidate factors were established. Exogenous Sprouty-2 and PEDF were both found to decrease blood vessel density in the wound bed during the proliferative phase, while inhibition of endogenous PEDF delayed vessel regression during the remodeling phase. Exogenous PEDF further promoted vascular integrity and increased collagen maturity. These results demonstrated that Sprouty-2 and PEDF contribute to the resolution of healing wounds by causing regression of immature blood vessels, thus promoting a return to tissue homeostasis after injury. In this Thesis, we also introduced a novel systems biology approach to the study of the complex mechanisms of healing, particularly angiogenesis and ECM-remodeling. High-throughput, time-course gene expression data of skin and oral mucosal wounds was analyzed via biological network analysis in the Angiome, Matrisome and combined networks. Results demonstrated significant co-regulation between angiogenesis and ECM-remodeling, with regulatory differences observed in oral versus skin wounds. Systems methods efficiently combined database knowledge with high-throughput experimental data to approach comprehensive data-driven models of healing wounds.