Regulation of Osteoblasts by ACTH and VEGF

ACTH 和 VEGF 对成骨细胞的调节

• 批准号: 10177859
• 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/10177859
• 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.

我们正在研究因糖皮质激素（GC）导致的成骨细胞死亡如何被血管抵消 内皮生长因子（VEGF）。开发成骨细胞表达ACTH受体和成骨细胞 响应肾上腺皮质激素（ACTH）的响应。连续立体治疗减少 ACTH的产量为低水平。在具有大剂量GC的兔子中，我们表明间歇性ACTH最少 剂量升高ACTH四个小时，截至骨分子症降低。我们最近的研究表明成骨细胞 VEGF增加了生长和分化。此外，ACTH是调节VEGF的几个因素之一 骨骼产生。在骨骼中，就像在肾上腺一样，ACTH的作用很复杂，而且系统的工作 将需要确定ACTH，VEGF和其他调节途径如何在骨骼中相互作用。 我们的假设是ACTH是骨骼快速骨骼生长和存活的主要调节剂 营业额，例如股骨头小梁骨。计划的工作将发现定量的ACTH剂量， 目前未知，会增加骨骼质量。计划的工作我们将使用兔子动物模型和人类 细胞确保与人类疾病相关。我们使用鼠标的工作在建模人类时遇到困难 骨反应；目前，最好的骨坏死动物模型是兔子。 理解的差距包括ACTH在骨细胞中的下游作用。 ACTH与 其他调节VEGF的系统，由炎症细胞，缺氧和其他细胞因子介导。这是 尚不知道骨骼在体内的反应如何通过ACTH给药的剂量或间隔的剂量或间隔。 特定的目标1将使用乙酸甲酯（MPA）处理的兔子来定义依赖性 VEGF合成，ACTH浓度和剂量间隔的骨坏死。具体来说，我们将定义 ACTH效应的浓度依赖性。我们将研究对不同ACTH的VEGF产生的影响 注射，相对于单独或未经处理的兔子的仓库MPA。 ACTH将每天在上午8点，0.01注射 0.03、0.1和0.3 µg/kg，持续28天。骨坏死，骨转换，血清ACTH和皮质类固醇将是 测量。这将确定特定ACTH剂量与抑制骨坏死的关系。 此外，为了确定ACTH给药频率对有效性的影响，我们将比较 每天两次或0.1或0.1或0.3 µg/kg，ACTH为0.05或0.15 µg/kg。 具体目标2将研究体外成骨细胞对ACTH和VEGF的反应机理。 为了确定ACTH除了VEGF之外是否还提供生存信号，我们将研究 成骨细胞可为VEGF，带有和不带有ACTH。还将测量细胞增殖和基质合成 作为正常和低氧条件下成骨细胞的调节细胞因子的产生。进一步定义 VEGF响应，我们将消除使用VEGF受体-1和-2（FLT -1和FLK -1）的成骨细胞。这会 在没有自分泌VEGF响应的情况下定义ACTH效应。 特定的目标3将决定ACTH如何调节糖皮质激素治疗的人的VEGF产生 细胞。这将包括评估免疫细胞通过成骨细胞调节VEGF产生的程度。 为此，我们将制作混合培养物，包括巨噬细胞或T淋巴细胞。我们将描述VEGF 这些培养物的生产，并确定VEGF调节是否涉及特定的细胞因子及其 对糖皮质激素的敏感性。此外，我们将确定ACTH合成是否发生在骨中 有意义的数量。特别是，我们将分析促蛋白酶皮质激素（POMC）的表达和处理 在包括成骨细胞，淋巴细胞和巨噬细胞在内的骨细胞中。 这项工作将使用创新方法定义骨骼中新的代谢调节途径。它会掉下来 关于有助于骨坏死的机制的新启示，这是一个严重而常见的问题。