ISOLATION OF THE SERUM FACTOR RESPONSIBLE FOR PSEUDOXANTHOMA ELASTICUM

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objectives. The long-term goal of this study is to better understand the remarkable biology and structural organization of elastic fibers. This general goal is best achieved by studying these fibers in vitro and in vivo using both human and/or animal models. The specific goal of our project is to identify and characterize the molecular events leading to elastic fiber alterations and calcification in pseudoxanthoma elasticum (PXE) as a pertinent disease model that should help elucidate the dynamics governing the homeostasis of elastic fiber proteins. Specific Aim 1: Determine the defect in the assembly of elastic fiber caused by PXE serum in vitro. We will use primary cell lines of skin fibroblasts and normal aorta smooth muscle cells with culture medium supplemented with serum from PXE patients or normal individuals. The principal elastic fiber components (elastin, fibulin-4, -5, fibrillin-1, -2, MAGP1, LOX and LOXL) will be examined by immunohistology and quantified during various stages of elastic fiber deposition, from pre- to post-confluence. Structural alterations will be evaluated by electron microscopy. The production of elastic fiber-specific proteases by fibroblasts and smooth muscle cells in the presence of PXE serum will also be evaluated by zymography. Specific Aim 2: Isolate serum factor(s) interfering with elastic fiber formation. The nature of the PXE serum factor(s) is unknown. We propose to determine the molecular characteristics of the PXE serum active component(s) and establish an isolation protocol based on chromatographic fractionations followed by mass spectroscopy analysis. The fractions will be tested using in vitro functional assays.