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. ? ?
