Regulation of Osteoblasts by ACTH and VEGF

我们正在研究糖皮质激素(GC)引起的成骨细胞死亡是如何被血管所抵消的。 血管内皮细胞生长因子(VEGF)。发育中的成骨细胞表达ACTH受体，成骨细胞 表达促肾上腺皮质激素(ACTH)反应的血管内皮生长因子。持续的类固醇治疗可以减少 ACTH产量降至较低水平。在大剂量GC的兔中，我们发现间歇性ACTH至少 连续4小时升高ACTH的剂量大大减少了骨坏死。我们最近的研究表明，成骨细胞 血管内皮生长因子可促进细胞生长和分化。此外，促肾上腺皮质激素是调节血管内皮生长因子的几个因素之一。 在骨骼方面的生产。因此，在骨骼中，就像在肾上腺中一样，促肾上腺皮质激素的作用是复杂的、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通路如何在骨骼中相互作用。 我们的假设是，促肾上腺皮质激素是骨骼快速生长和存活的主要调节因素。 翻转股骨头等松质骨。计划中的工作将找到ACTH的定量剂量， 目前尚不清楚，这会增加骨量。工作计划我们将使用兔子动物模型，和人类 确保与人类疾病相关的细胞。我们用老鼠为人类建模时遇到了困难 骨反应：目前最好的骨坏死动物模型是兔。 理解上的差距包括骨细胞中ACTH的下游作用。促肾上腺皮质激素与神经生长因子的相互作用 其他调节血管内皮生长因子的系统，由炎症细胞、缺氧和额外的细胞因子介导。它是 尚不清楚体内骨骼的反应如何随ACTH给药的剂量或间隔而变化。 特定目标1将使用甲基强的松龙(MPA)处理的兔来定义对 骨坏死对血管内皮生长因子合成、ACTH浓度和剂量间隔的影响。具体地说，我们将定义 ACTH效应的浓度依赖性。我们将研究不同促肾上腺皮质激素对血管内皮生长因子产生的影响 注射，相对于单独或未经处理的兔体内的甲孕酮。每天早上8点，0.01点注射ACTH， 0.03、0.1和0.3微克/公斤，共28天。骨坏死、骨转换、血清ACTH和皮质类固醇 量过了。这将建立特定的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每天两次服用0.05或0.15微克/公斤，而每天一次服用0.1或0.3微克/公斤。 目的2体外研究人成骨细胞对ACTH和血管内皮生长因子的反应机制。 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 成骨细胞向血管内皮生长因子转化，有无促肾上腺皮质激素。还将测量细胞增殖和基质合成。 在正常和低氧条件下，成骨细胞产生调节性细胞因子。进一步定义 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(Flt-1和Flk-1)的成骨细胞被清除。这将是 允许在没有自分泌血管内皮生长因子反应的情况下定义ACTH效应。 特异性目标3将确定ACTH如何调节糖皮质激素治疗的人血管内皮生长因子的产生 细胞。这将包括评估免疫细胞在多大程度上调节成骨细胞产生血管内皮生长因子。 为此，我们将进行包括巨噬细胞或T淋巴细胞在内的混合培养。我们将描述血管内皮细胞生长因子 在这些培养物中产生，并确定血管内皮生长因子的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 有意义的数额。具体地说，我们将分析前阿片黑色素皮质激素(POMC)的表达和加工 在骨细胞中，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，在骨骼中定义一种新的代谢调节途径。它会掉下来的 对导致骨坏死的机制有了新的认识，骨坏死是一个严重而普遍的问题。

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.