Regulation of Osteoblasts by ACTH and VEGF

ACTH 和 VEGF 对成骨细胞的调节

• 批准号: 8815885
• 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 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8815885
• 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; Dependency; Diabetes Mellitus; Differentiation and Growth; Dose; Event; Feedback; Figs - dietary; Frequencies; Glucocorticoids; Growth; Head; Hour; Human; Hypoxia; Immune; In Vitro; Inflammatory; Injection of therapeutic agent; Insulin; Knockout Mice; Light; Lymphocyte; Measures; Mediating; Mesenchymal Stem Cells; Metabolic; Methods; Methylprednisolone; Modeling; Mus; Oryctolagus cuniculus; Osteoblasts; Oxygen; Oxygen measurement, partial pressure, arterial; Pilot Projects; Pituitary Gland; Prevention; Process; Production; Regulation; Regulatory Pathway; Relative (related person); Serum; Severities; Signal Transduction; Steroids; System; Time; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Veterans; Work; autocrine; bone; bone cell; bone turnover; capillary; 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）诱导的骨坏死的初始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，不是直接由GC引起的。骨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，在高转换骨如股骨头松质骨中突出，表达促肾上腺皮质激素（ACTH）受体。我们发现，成骨细胞表达VEGF强烈响应促肾上腺皮质激素。持续的类固醇治疗可将ACTH降低到非常低的水平。在高剂量GC的兔子中，我们发现间歇性给予ACTH大大减少了骨坏死。我们的假设是，ACTH是骨生长和存活的关键调节因子，特别是在骨转换迅速的区域。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增加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，所以需要系统的研究来确定ACTH、VEGF和其他调节途径在骨中如何相互作用。 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。这些相互作用可能由炎性细胞、缺氧或其他细胞信号介导。目前尚不清楚促肾上腺皮质激素的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给药的频率或剂量而变化。目的1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。为了确定促肾上腺皮质激素是否提供生存信号，除了VEGF，我们将研究成骨细胞对VEGF的反应，有和没有促肾上腺皮质激素。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。为了确定VEGF的反应，我们将使成骨细胞与VEGF受体-1和-2（ft-1和fk-1）消除。这将允许ACTH对成骨细胞的作用在没有自分泌VEGF反应的情况下被定义。目的2将确定促肾上腺皮质激素如何调节血管内皮生长因子的生产，在GC处理的骨。为了确定免疫细胞如何调节成骨细胞产生VEGF，我们将进行包括巨噬细胞或淋巴细胞的混合培养。在 此外，将研究在正常GC和致密骨下具有升高的ACTH的Scarb 1小鼠模型，以确定体内骨和脉管系统的变化。我们将描述培养中VEGF的产生，并确定VEGF的调节是否涉及组织培养和小鼠中的特异性细胞因子。为了确定缺氧的影响，我们将比较人细胞培养物中7%与20%氧气中VEGF的产生。分析将包括作为氧张力的函数的关键细胞因子的产生。为了确定促肾上腺皮质激素合成是否发生在骨，我们将分析前阿黑皮素（POMC）的表达和加工成骨细胞，淋巴细胞和巨噬细胞。目的3用长效醋酸甲泼尼龙（MPA）治疗家兔，确定骨内VEGF合成对ACTH浓度和给药间隔的依赖性。家兔是CG诱导的骨坏死的最佳体内模型;将发生类固醇糖尿病，但将用胰岛素控制。相对于单独的贮库型MPA或无治疗，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。为了确定给药频率对疗效的影响，我们将比较ACTH 0.05 kg和0.15 kg每日两次与0.1或0.3 kg每日一次的效果。这项工作将使用创新的方法来定义一个新的骨代谢调控途径。它将建立新的机制，有助于骨坏死，这可能会减少其发生。