We are studying how osteoblast death due to glucocorticoids (GCs) is counteracted by vascular endothelial growth factor (VEGF). Developing osteoblasts express the ACTH receptor, and osteoblasts express VEGF in response to the adrenocorticotropic hormone (ACTH). Continuous steroid treatment reduces ACTH production to low levels. In rabbits with high-dose GC, we showed that intermittent ACTH at minimum doses to elevate ACTH for four hours greatly reduced osteonecrosis. Our recent studies show that osteoblast growth and differentiation is increased by VEGF. Further, ACTH is one of several factors that regulate VEGF production in bone. Thus, in bone, as in the adrenal, the actions of ACTH are complex, and systematic work will be needed to determine how ACTH, VEGF, and other regulatory pathways interact in bone. Our hypothesis is that ACTH is a major regulator of bone growth and survival in regions with rapid bone turnover such as femoral head trabecular bone. The work planned will find quantitative ACTH doses, currently unknown, that increase bone mass. Work planned we will use a rabbit animal model, and human cells to assure relevancy to human disease. Our work using mice encountered difficulties in modeling human bone response; at present the best animal model for osteonecrosis is the rabbit. Gaps in understanding include downstream actions of ACTH in bone cells. Interactions of ACTH with other systems that regulate VEGF, mediated by inflammatory cells, hypoxia, and by additional cytokines. It is not known how response of bone in vivo varies with the dose or interval of ACTH administration. Specific Aim 1 will use methylprednisolone acetate (MPA)-treated rabbits to define the dependency of osteonecrosis on VEGF synthesis, ACTH concentration, and dose interval. Specifically, we will define concentration dependency of ACTH effects. We will study effects on VEGF production of varying ACTH injection, relative to depot MPA alone or in untreated rabbits. ACTH will be injected daily, at 8 AM, at 0.01, 0.03, 0.1, and 0.3 µg/kg, for 28 days. Osteonecrosis, bone turnover, serum ACTH and corticosteroids will be measured. This will establish the relationship of specific ACTH doses to suppression of osteonecrosis. Additionally, to define effect of frequency of ACTH administration on efficacy, we will compare the effects of ACTH at 0.05 or 0.15 µg/kg twice daily versus 0.1 or 0.3 µg/kg once daily. Specific Aim 2 will study the mechanism of response of human osteoblasts to ACTH and VEGF in vitro. To determine whether ACTH provides survival signals in addition to VEGF, we will study the response of osteoblasts to VEGF, with and without ACTH. Cell proliferation and matrix synthesis will be measured, as well as production of regulatory cytokines by osteoblasts under normal and hypoxic conditions. Further to define the VEGF response, we will make osteoblasts with VEGF receptors -1 and -2 (flt-1 and flk-1) eliminated. This will allow ACTH effects to be defined in ...