HOMOCYSTEINE MODIFIES BONE MATRIX AND MINERALIZATION

描述（由申请人提供）：几年前，我们假设中等水平的高同型半胱氨酸血症可能在许多结缔组织病理中起因果作用，这些病理通常被认为是衰老的正常后果。我们强调了同型半胱氨酸尿的标志性表现（血清同型半胱氨酸浓度通常在100 - 100 （m/L）之间，即闭塞性血管疾病、骨质疏松症、精神退化和异位性晶状体）与“正常”衰老的对应表现（血清同型半胱氨酸浓度在10 - 100 （m/L）之间，即闭塞性血管疾病、骨质疏松症、痴呆和老花眼之间）之间的相似之处。重要的是要认识到，这些高同型半胱氨酸（Hcy）的表现通常是浓度和年龄依赖性的，即，高同型半胱氨酸血症越严重，发病年龄越小。例如，在未治疗的同型半胱氨酸尿患者中，骨质疏松症在15岁时可被x线片检测到的比例为40%，在30岁时可被检测到的比例为80%。相反，中度高同型半胱氨酸血症需要较长时间的暴露才能产生类似的病理。因此，就骨骼而言，我们假设长时间、中度升高的同型半胱氨酸在与衰老相关的骨质疏松症的发展中起着因果作用。在这项发展资助中，我们寻求资金，为我们主要假设的一些假设建立实验证明。我们假设1)Hcy和/或Hcytl介导的骨细胞外基质蛋白的改变发生在体内，2)Hcy和/或Hcytl可以在体外破坏成骨细胞的骨形成。为了验证这些假设，我们制定了以下各自的具体目标：1)在本应用中，我们寻求从人骨中分离出经Hcy或Hcytl修饰的蛋白质，并在可能的情况下对其进行鉴定。我们将检查皮质骨和松质骨，并试图确定两种类型的人骨中蛋白相关Hcy之间是否存在与年龄相关的关联。为了更好地理解这种翻译后修饰的化学作用，我们还将实验研究导致Hcy掺入骨基质蛋白的条件。2)建立人骨髓基质细胞培养，诱导成骨细胞分化，评估Hcy和Hcytl升高对细胞增殖率、细胞外基质形成、成骨细胞分化程度和基质矿化的影响。我们还将研究Hcy或Hcytl改变预形成成骨细胞衍生基质矿化程度的能力。这些研究应该为骨质疏松的可能机制提供新的数据，并提出新的治疗和预防方法。

DESCRIPTION (provided by applicant): We hypothesized several years ago that moderate levels of hyperhomocysteinemia may have a causal role in a number of connective tissue pathologies typically thought of as normal consequences of aging. We emphasized the parallels between the hallmark manifestations of homocystinuria (with serum homocysteine concentrations typically >100 (m/L), ie, occlusive vascular disease, osteoporosis, mental deterioration, and ectopia lentis, and the counterpart manifestations of "normal" aging (with serum homocysteine concentrations between 10 and 100 (m/L), ie, occlusive vascular disease, osteoporosis, dementia, and presbyopia. It is important to recognize that these manifestations of elevated homocysteine (Hcy) are generally concentration and age-dependent, i.e., the more severe the hyperhomocysteinemia, the younger the age of onset. For example, osteoporosis is detectable by radiographs in 40% of untreated homocystinuric patients by age 15 and in 80% by age 30. Conversely, moderate hyperhomocysteinemia should require prolonged periods of exposure to produce similar pathology. Thus, with regard to bone, we hypothesize that prolonged, moderately elevatated homocysteine has a causal role in the development of the osteoporosis associated with aging. In this developmental grant we seek funding to establish experimental proof of principle for some of the assumptions underlying our main hypothesis. We HYPOTHESIZE that 1) Hcy- and/or Hcytl-mediated alterations of bone extracellular matrix proteins occur in vivo, and that 2) Hcy and/or Hcytl can impair bone formation by osteoblasts in vitro. To test these hypotheses, we have developed the following respective SPECIFIC AIMS: 1) In this application we seek to isolate proteins from human bone that are modified with Hcy or Hcytl and identify them, if possible. We will examine both cortical and cancellous bone and seek to determine if there is an age-related association between protein-associated Hcy in both types of human bone. In order to better understand the chemistry of this post- translational modification, we will also experiment investigate conditions that result in the incorporation of Hcy into bone matrix proteins. 2) We will establish human bone marrow stromal cell cultures, induce osteoblast differentiation, and evaluate the effects of elevated Hcy and Hcytl on rates of cell proliferation, extracellular matrix formation, extent of osteoblast differentiation and matrix mineralization. We shall also examine the ability of Hcy or Hcytl to alter the extent of mineralization of pre-formed osteoblast-derived matrices. These studies should provide new data regarding possible mechanisms of osteoporosis as well as suggest new therapies for treatment and prevention.