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，至少在很大程度上，不是由骨生长和骨坏死直接引起的。生存受到许多系统的调节，血管内皮生长因子（VEGF）对于成骨细胞的发育至关重要，尤其是在股骨头等高周转骨骼中。我们发现，在接受高剂量 GC 的兔子中，连续使用类固醇治疗后，成骨细胞会强烈地表达 VEGF，但间歇性给予 ACTH 后，其水平会大大降低。我们的假设是促肾上腺皮质激素是骨生长和存活的关键调节剂，特别是在骨转换快速的区域。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，因为 ACTH 是调节骨中 VEGF 产生的几个因素之一，因此需要进行系统研究来确定 ACTH、VEGF 和其他调节剂的作用。通路在骨中相互作用。 理解上的差距包括对 ACTH 在骨细胞中的下游作用知之甚少，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。 ACTH 合成发生在骨骼中。目前尚不清楚 ACTH 在骨骼中的作用如何随 ACTH 给药的频率或剂量而变化。目的 1 将研究成骨细胞对 ACTH 和 VEGF 的反应机制，以确保与人类的相关性。对于体外的人类细胞，除了 VEGF 之外，我们还将研究成骨细胞对 VEGF 的反应，并测量细胞增殖和基质合成，以及 VEGF 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，这将使 ACTH 对成骨细胞产生影响。在没有自分泌 VEGF 反应的情况下进行定义 目标 2 将确定 ACTH 如何调节 GC 处理的骨骼中的 VEGF 产生。 为了确定免疫细胞如何调节成骨细胞的 VEGF 产生，我们将进行包括巨噬细胞或淋巴细胞在内的混合培养。 此外，我们将研究 Scarb1 小鼠模型，该模型在正常 GC 和致密骨中 ACTH 升高，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% 氧气的 VEGF 产生，分析将包括关键细胞因子的产生作为氧张力的函数。无论 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) 确定骨骼中 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。相对于单独的长效 MPA 或不治疗，不同 IV ACTH 注射的 VEGF 产生将建立浓度依赖性，每天上午 8 点以 0.01 至 0.3 mg/kg 注射，持续 28 天。将测量血清 ACTH 和皮质类固醇，这将建立 ACTH 骨坏死抑制的剂量依赖性，为了确定给药频率对疗效的影响，我们将比较其效果。 ACTH 0.05 µkg 和 0.15 µkg 每天两次，相对于 0.1 或 0.3 µkg 每天一次。这项工作将使用创新方法来定义骨骼中的新代谢调节途径，它将建立有助于骨坏死的新机制。它的发生。