The Role of Osteopontin in Postnatal Vascular Growth: Functional Effects in Ischemic Limb Collateral Vessel Formation and Long Bone Fracture Healing
Duvall, Craig Lewis
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Postnatal vascular growth is a complex process involving multiple cells types whose functionality is orchestrated by a variety of soluble extracellular growth factors, mechanical stimuli, and matrix derived cues. The central goal for this dissertation project was to elucidate the role of osteopontin, a non-collagenous extracellular matrix protein, in postnatal vascular growth. At the onset, we concluded that the current methods for measurement of vascularity in small animal models were lacking. To address this shortcoming, we pursued micro-CT imaging for analysis of three-dimensional blood vessel architecture. We were able to demonstrate that micro-CT imaging provides an objective, quantitative, and three-dimensional methodology for evaluation of vascular networks that has broad applicability to preclinical studies. Next, we sought to apply the developed imaging techniques, along with other complementary methodologies, to explore the role of osteopontin in postnatal vascular growth. Osteopontin was previously known to elicit survival, migration, and other relevant activities in multiple cell types involved in postnatal vascular growth. Therefore, we sought to determine the in vivo significance of osteopontin in this process. To do so, we compared wild type and Osteopontin-/- mice for (1) their ability to form collateral vessels and functionally recover following acute induction of hind limb ischemia and (2) their capacity for neovascularization, mineralization, remodeling, and the restoration of mechanical properties during fracture healing. Data suggested that OPN is a critical regulator of collateral vessel formation and that this effect is driven by its role in mediating monocyte/macrophage migration and functionality. Secondly, we found that the presence of osteopontin was essential for normal early callus formation, neovascularization, late stage callus remodeling, and restoration of biomechanical strength. Abnormal collagen organization was observed within the remodeling fractures of Osteopontin-/- mice, and we hypothesize that a unifying link between the vascular and bone defects may be related to deficient matrix organization and remodeling. In conclusion, the imaging techniques developed in this thesis provide a novel methodology for quantitative analysis of vascular structures in small animal models. Secondly, this project has yielded an improved understanding of the basic pathophysiological mechanisms that control postnatal blood vessel growth and bone fracture healing.