Regulation of Osteoblasts by ACTH and VEGF

ACTH 和 VEGF 对成骨细胞的调节

• 批准号: 10001755
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001755
• 关键词: Acetates; Adrenal Cortex Hormones; Adrenal Glands; Animal Model; Blood Vessels; Blood capillaries; Bone Growth; Bone necrosis; Cell Culture Techniques; Cell Hypoxia; Cell Proliferation; Cells; Cessation of life; Circadian Rhythms; Clinical; Complex; Corticotropin; Corticotropin Receptors; Dependence; Differentiation and Growth; Dose; Endothelial Growth Factors Receptor; Feedback; Frequencies; Glucocorticoids; Head; Hormones; Hour; Human; Hypoxia; Immune; In Vitro; Inflammatory; Injections; Light; Lymphocyte; Measures; Mediating; Mesenchymal Stem Cells; Metabolic; Methods; Methylprednisolone; Modeling; Mus; Oryctolagus cuniculus; Osteoblasts; PF4 Gene; Pituitary Gland; Production; Regulation; Regulatory Pathway; Serum; Signal Transduction; Steroids; System; T-Lymphocyte; Time; Tissues; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Work; autocrine; bone; bone cell; bone mass; bone turnover; cytokine; human disease; human model; in vivo; innovation; macrophage; novel; prevent; response; substantia spongiosa; tissue culture

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 the absence of autocrine VEGF response. Specific Aim 3 will determine how ACTH modulates VEGF production in glucocorticoid-treated human cells. This will include evaluating the extent to which immune cells regulate production of VEGF by osteoblasts. To do this, we will make mixed cultures including macrophages or T-lymphocytes. We will characterize VEGF production in these cultures, and determine whether VEGF regulation involves specific cytokines, and its sensitivity to glucocorticoids. In addition, we will determine whether ACTH synthesis occurs in bone in meaningful amounts. Specifically, we will analyze pro-opiomelanocorticoid (POMC) expression and processing in bone cells including osteoblasts, lymphocytes and macrophages. This work will define a new metabolic regulatory pathway in bone, using innovative methods. It will shed new light on mechanisms that contribute to osteonecrosis, which is a serious and common problem.

我们正在研究糖皮质激素（GCs）引起的成骨细胞死亡如何被血管内皮细胞抵消， 内皮生长因子（VEGF）。发育中的成骨细胞表达ACTH受体， 表达VEGF以响应促肾上腺皮质激素（ACTH）。持续类固醇治疗可减少 促肾上腺皮质激素的生产水平低。在用高剂量GC的兔子中，我们发现间歇性ACTH在最低限度， 剂量升高促肾上腺皮质激素4小时大大减少了骨坏死。我们最近的研究表明成骨细胞 生长和分化通过VEGF增加。此外，ACTH是调节VEGF的几种因子之一 在骨头里生产。因此，在骨，在肾上腺，行动的促肾上腺皮质激素是复杂的，系统的工作 将需要确定促肾上腺皮质激素，血管内皮生长因子，和其他调节途径在骨中相互作用。 我们的假设是，ACTH是骨生长和生存在快速骨生长区域的主要调节因子。 如股骨头骨小梁的周转。这项计划的工作将找到定量的ACTH剂量， 目前尚不清楚，可以增加骨量。工作计划，我们将使用兔子动物模型，和人类 细胞，以确保与人类疾病的相关性。我们用小鼠的工作在模拟人类时遇到了困难 骨反应;目前骨坏死的最佳动物模型是兔。 理解上的差距包括骨细胞中ACTH的下游作用。ACTH与 其他调节VEGF的系统，由炎性细胞、缺氧和其他细胞因子介导。是 不知道体内骨的反应如何随ACTH给药的剂量或间隔而变化。 具体目标1将使用醋酸甲泼尼龙（MPA）治疗的家兔来确定 骨坏死对VEGF合成、ACTH浓度和给药间隔的影响。具体来说，我们将定义 ACTH效应的浓度依赖性。我们将研究不同的ACTH对VEGF产生的影响， 注射，相对于单独的贮库型MPA或在未处理的兔中。ACTH将每天注射，上午8点，0.01， 0.03、0.1和0.3 µg/kg，持续28天。骨坏死、骨转换、血清促肾上腺皮质激素和皮质类固醇将 测定了这将建立特定ACTH剂量与抑制骨坏死的关系。 此外，为了确定ACTH给药频率对疗效的影响，我们将比较 ACTH，0.05或0.15 µg/kg，每日两次，对比0.1或0.3 µg/kg，每日一次。 具体目标2研究人成骨细胞对ACTH和VEGF的反应机制。 为了确定除了VEGF外，ACTH是否提供生存信号，我们将研究 成骨细胞对VEGF的影响，有和没有ACTH。细胞增殖和基质合成也将被测量， 如成骨细胞在正常和缺氧条件下产生调节性细胞因子。为了进一步定义 VEGF的反应，我们将使成骨细胞与VEGF受体-1和-2（flt-1和flk-1）消除。这将 允许在没有自分泌VEGF反应的情况下定义ACTH作用。 特异性目的3将确定ACTH如何调节糖皮质激素治疗的人VEGF的产生。 细胞这将包括评估免疫细胞调节成骨细胞产生VEGF的程度。 为此，我们将进行包括巨噬细胞或T淋巴细胞的混合培养。我们将描述VEGF 在这些培养物中的产生，并确定VEGF调节是否涉及特定的细胞因子，及其 对糖皮质激素敏感。此外，我们将确定ACTH合成是否发生在骨骼中， 有意义的金额。具体来说，我们将分析前阿黑皮素（POMC）的表达和加工 包括成骨细胞、淋巴细胞和巨噬细胞。 这项工作将使用创新的方法定义一种新的骨代谢调节途径。它会脱落 新的光的机制，有助于骨坏死，这是一个严重的和常见的问题。