Regulation of Osteoblasts by ACTH and VEGF

ACTH 和 VEGF 对成骨细胞的调节

• 批准号: 9788189
• 负责人: Harry C. Blair
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788189
• 关键词: Acetates; Adrenal Cortex Hormones; Adrenal Glands; Ally; Animal Model; Biological Assay; Blood Vessels; Blood capillaries; Bone Growth; Bone necrosis; Cell Culture Techniques; Cell Hypoxia; Cell Proliferation; Cells; Cessation of life; Circadian Rhythms; Complex; Corticotropin; Corticotropin Receptors; Data; Dependence; Diabetes Mellitus; Differentiation and Growth; Dose; Endothelial Growth Factors Receptor; Event; Feedback; Frequencies; Glucocorticoids; Growth; Head; Hour; Human; Hypoxia; Immune; In Vitro; Inflammatory; Injections; Insulin; Knockout Mice; Light; Lymphocyte; Measures; Mediating; Mesenchymal Stem Cells; Metabolic; Methods; Methylprednisolone; Modeling; Mus; Oryctolagus cuniculus; Osteoblasts; Oxygen; Pilot Projects; Pituitary Gland; Prevention; Production; Regulation; Regulatory Pathway; Serum; Severities; Signal Transduction; Steroids; System; Time; VEGFA gene; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Veterans; Work; autocrine; bone; bone cell; bone turnover; cytokine; hip replacement arthroplasty; human disease; in vivo; in vivo Model; innovation; macrophage; mouse model; novel; public health relevance; response; substantia spongiosa; tissue culture

DESCRIPTION (provided by applicant): The initial event in glucocorticoid (GC)-induced osteonecrosis, in many cases, is death of bone-synthesizing osteoblasts. Our preliminary work shows that this osteoblast death, at least in major part, is not directly caused by the GC. Bone growth and survival is regulated by many systems, vascular endothelial growth factor (VEGF) being of critical importance. Developing osteoblasts, prominent in in high turnover bone such as femoral head trabecular bone, express the adrenocorticotropic hormone (ACTH) receptor. We discovered that osteoblasts express VEGF strongly in response to ACTH. Continuous steroid treatment reduces ACTH to very low levels. In rabbits with high-dose GC, we showed that intermittent administration of ACTH greatly reduced osteonecrosis. Our hypothesis is that ACTH is a key regulator of bone growth and survival, particularly in regions with rapid bone turnover. However, mesenchymal stem cells or osteoblasts express VEGF receptors and ACTH receptors. Further, our pilot studies show that VEGF increases growth and differentiation or osteoblasts. Because ACTH is one of several factors that regulate VEGF production in bone, systematic study is needed to determine how ACTH, VEGF, and other regulatory pathways interact in bone. Gaps in understanding include that downstream actions of ACTH in bone cells are poorly understood. The interactions of ACTH with other systems that regulate VEGF are unclear. These interactions may be mediated by inflammatory cells, hypoxia, or additional cell signals. It is not known whether ACTH synthesis occurs in bone. It is not known how ACTH action in bone varies with frequency or dose of ACTH administration. Aim 1 will study the mechanism of response of osteoblasts to ACTH and VEGF. To assure relevancy to human disease, study will include human cells 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. To define the VEGF response, we will make osteoblasts with VEGF receptors -1 and -2 (ft-1 and fk-1) eliminated. This will allow ACTH effects on osteoblasts to be defined in the absence of autocrine VEGF response. Aim 2 will determine how ACTH modulates VEGF production in GC-treated bone. To determine how immune cells regulate production of VEGF by osteoblasts, we will make mixed cultures including macrophages or lymphocytes. In addition, the Scarb1 mouse model, which has elevated ACTH at normal GC and dense bone, will be studied to determine changes to bone and vasculature in vivo. We will characterize VEGF production in culture, and determine whether VEGF regulation involves specifc cytokines, in tissue cultures and in mice. To establish the effect of hypoxia, we will compare VEGF production in 7% versus 20% oxygen in human cell cultures. Analysis will include production of key cytokines as a function of oxygen tension. To determine whether ACTH synthesis occurs in bone, we will analyze pro-opiomelanocorticoid (POMC) expression and processing in osteoblasts, lymphocytes and macrophages. Aim 3 will defne the dependency of VEGF synthesis in bone on ACTH concentration and dose interval using depot methylprednisolone acetate (MPA)-treated rabbits. Rabbits are the best in vivo model for CG-induced osteonecrosis; steroid diabetes will occur but will be controlled with insulin. Effects on VEGF production of varying IV ACTH injection, relative to depot MPA alone or no treatment, will establish concentration dependency. ACTH will be injected daily, at 8 AM, at 0.01 to 0.3 mg/kg, for 28 days. Osteonecrosis, bone turnover, serum ACTH and corticosteroids will be measured. This will establish the dose dependency of ACTH osteonecrosis suppression. To defne effect of frequency of administration on efficacy, we will compare the effects of ACTH 0.05 �kg and 0.15 �kg twice daily versus 0.1 or 0.3 �kg once daily. This work will use innovative methods to defne a novel metabolic regulatory pathway in bone. It will establish new mechanisms that contribute to osteonecrosis, which may allow reduction of its occurrence.

描述（由申请人提供）： 在许多情况下，糖皮质激素（GC）引起的骨坏死的最初事件是骨合成成骨细胞的死亡。我们的初步工作表明，这种成骨细胞死亡，至少大部分，并不是直接由GC引起的。骨骼生长和存活受到许多系统的调节，其中血管内皮生长因子 (VEGF) 至关重要。发育中的成骨细胞，在股骨头小梁骨等高周转骨中突出，表达促肾上腺皮质激素（ACTH）受体。我们发现成骨细胞强烈表达 VEGF，以响应 ACTH。持续类固醇治疗可将 ACTH 降低至非常低的水平。在接受高剂量 GC 的兔子中，我们发现间歇性给予 ACTH 可以大大减少骨坏死。我们的假设是促肾上腺皮质激素是骨骼生长和存活的关键调节剂，特别是在骨转换快速的区域。然而，间充质干细胞或成骨细胞表达VEGF受体和ACTH受体。此外，我们的初步研究表明 VEGF 可以促进成骨细胞的生长和分化。由于 ACTH 是调节骨中 VEGF 产生的几个因素之一，因此需要系统研究来确定 ACTH、VEGF 和其他调节途径如何在骨中相互作用。 理解上的差距包括对骨细胞中 ACTH 的下游作用知之甚少。 ACTH 与其他调节 VEGF 的系统的相互作用尚不清楚。这些相互作用可能是由炎症细胞、缺氧或其他细胞信号介导的。目前尚不清楚 ACTH 合成是否发生在骨骼中。目前尚不清楚 ACTH 在骨中的作用如何随 ACTH 给药频率或剂量的变化而变化。目标1将研究成骨细胞对ACTH和VEGF的反应机制。为了确保与人类疾病的相关性，研究将包括体外人类细胞。为了确定除了 VEGF 之外，ACTH 是否还提供生存信号，我们将研究在有或没有 ACTH 的情况下成骨细胞对 VEGF 的反应。将测量细胞增殖和基质合成，以及成骨细胞调节细胞因子的产生。为了定义 VEGF 反应，我们将消除 VEGF 受体 -1 和 -2（ft-1 和 fk-1）的成骨细胞。这将允许在没有自分泌 VEGF 反应的情况下定义 ACTH 对成骨细胞的影响。目标 2 将确定 ACTH 如何调节 GC 处理的骨骼中 VEGF 的产生。为了确定免疫细胞如何调节成骨细胞产生 VEGF，我们将制备包括巨噬细胞或淋巴细胞的混合培养物。在 此外，我们还将研究 Scarb1 小鼠模型，该模型在正常 GC 和致密骨中 ACTH 升高，将被研究以确定体内骨骼和脉管系统的变化。我们将表征培养物中 VEGF 的产生，并确定 VEGF 调节是否涉及组织培养物和小鼠中的特定细胞因子。为了确定缺氧的影响，我们将比较人类细胞培养物中 7% 和 20% 氧气条件下 VEGF 的产生。分析将包括关键细胞因子的产生作为氧张力的函数。为了确定 ACTH 合成是否发生在骨骼中，我们将分析阿片黑皮质激素原 (POMC) 在成骨细胞、淋巴细胞和巨噬细胞中的表达和加工。目标 3 将使用长效醋酸甲泼尼龙 (MPA) 治疗的兔子来定义骨中 VEGF 合成对 ACTH 浓度和剂量间隔的依赖性。兔子是CG诱导的骨坏死的最佳体内模型；类固醇糖尿病会发生，但可以通过胰岛素控制。相对于单独使用长效 MPA 或不进行治疗，不同 IV ACTH 注射液对 VEGF 产生的影响将建立浓度依赖性。每天上午 8 点以 0.01 至 0.3 mg/kg 注射 ACTH，持续 28 天。将测量骨坏死、骨转换、血清 ACTH 和皮质类固醇。这将建立 ACTH 骨坏死抑制的剂量依赖性。为了定义给药频率对疗效的影响，我们将比较每天两次 0.05 µkg 和 0.15 µkg ACTH 与每天一次 0.1 或 0.3 µkg 的效果。这项工作将使用创新方法来定义骨骼中新的代谢调节途径。它将建立有助于骨坏死的新机制，从而可能减少骨坏死的发生。