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)： 糖皮质激素(GC)诱导的骨坏死，在许多情况下，最初的事件是骨合成成骨细胞的死亡。我们的初步工作表明，这种成骨细胞的死亡，至少在很大程度上，不是由GC直接引起的。骨的生长和存活受多种系统的调控，其中血管内皮生长因子(VEGF)起着至关重要的作用。发育中的成骨细胞表达促肾上腺皮质激素(ACTH)受体，尤其是在高转换率的骨中，如股骨头小梁骨。我们发现成骨细胞强烈表达血管内皮生长因子作为对ACTH的反应。持续的类固醇治疗将ACTH降低到非常低的水平。在大剂量GC兔身上，我们发现间歇给药ACTH可显著减少骨坏死。我们的假设是，ACTH是骨生长和存活的关键调节因子，特别是在骨快速转换的区域。而间充质干细胞或成骨细胞表达血管内皮生长因子受体和促肾上腺皮质激素受体。此外，我们的初步研究表明，血管内皮生长因子可促进成骨细胞的生长和分化。由于ACTH是调节骨中血管内皮生长因子产生的几个因素之一，因此需要系统的研究来确定ACTH、血管内皮生长因子和其他调节通路如何在骨中相互作用。 理解上的差距包括对ACTH在骨细胞中的下游作用知之甚少。ACTH与其他调节血管内皮生长因子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、缺氧或额外的细胞信号介导的。目前尚不清楚ACTH的合成是否发生在骨骼中。目前尚不清楚ACTH在骨骼中的作用如何随ACTH给药频率或剂量的变化而变化。目的1研究成骨细胞对促肾上腺皮质激素和血管内皮生长因子的反应机制。为了确保与人类疾病的相关性，这项研究将包括体外培养的人类细胞。为了确定ACTH是否除了提供血管内皮生长因子外还提供生存信号，我们将研究成骨细胞在有和没有ACTH的情况下对血管内皮生长因子的反应。将测量细胞增殖和基质合成，以及成骨细胞产生的调节细胞因子。为了确定血管内皮生长因子的反应，我们将使带有血管内皮生长因子受体-1和-2(ft-1和Fk-1)的成骨细胞消失。这将允许在没有自分泌血管内皮生长因子反应的情况下确定ACTH对成骨细胞的影响。目的2将确定ACTH如何调节GC处理的骨中血管内皮生长因子的产生。为了确定免疫细胞如何调节成骨细胞产生血管内皮生长因子，我们将进行包括巨噬细胞或淋巴细胞的混合培养。在……里面 此外，在正常GC和致密骨中ACTH升高的Scar1小鼠模型将被研究，以确定体内骨骼和血管的变化。我们将表征培养中的血管内皮生长因子的产生，并确定在组织培养和小鼠中，血管内皮生长因子的调节是否涉及特定的细胞因子。为了确定缺氧的影响，我们将比较7%氧气和20%氧气对人类细胞培养中血管内皮生长因子的影响。分析将包括关键细胞因子的产生作为氧分压的函数。为了确定ACTH是否在骨中合成，我们将分析成骨细胞、淋巴细胞和巨噬细胞中阿片黑素皮质激素原(POMC)的表达和处理。目的3利用甲基强的松龙醋酸酯(MPA)处理兔，确定骨中血管内皮生长因子的合成对ACTH浓度和给药间隔的依赖性。兔是CG诱导的骨坏死的最好的活体模型；类固醇糖尿病会发生，但将通过胰岛素控制。不同静脉注射ACTH对血管内皮生长因子产生的影响，相对于单独或不处理甲孕酮，将建立浓度依赖关系。ACTH每天上午8点注射，剂量为0.01-0.3 mg/kg，共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抑制骨坏死的剂量依赖关系。为了明确给药频率对疗效的影响，我们将比较每天两次ACTH0.05�kg和0.15�kg与每天一次0.1或0.3�kg的效果。这项工作将使用创新的方法来定义骨中一种新的代谢调节途径。它将建立有助于骨坏死的新机制，这可能会减少骨坏死的发生。