ISOLATION OF THE SERUM FACTOR RESPONSIBLE FOR PSEUDOXANTHOMA ELASTICUM

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 目标。这项研究的长期目标是更好地了解弹性纤维非凡的生物学和结构组织。通过使用人类和/或动物模型在体外和体内研究这些纤维，可以最好地实现这一总体目标。我们项目的具体目标是识别和表征导致弹性假性黄瘤(PXE)弹性纤维改变和钙化的分子事件，作为一种相关的疾病模型，应该有助于阐明弹性纤维蛋白的动态平衡。 特异性目的1：体外检测PXE血清引起的弹性纤维组装缺陷。 我们将使用皮肤成纤维细胞和正常主动脉平滑肌细胞的原代细胞系，并在培养液中添加PXE患者或正常人的血清。主要的弹性纤维成分(弹性蛋白、纤维蛋白-4、-5、纤维蛋白-1、-2、MAGP1、LOX和LOXL)将在弹性纤维沉积的不同阶段(从融合前到融合后)进行免疫组织学检测和定量。结构变化将通过电子显微镜进行评估。在PXE血清存在的情况下，成纤维细胞和平滑肌细胞产生的弹性纤维特定的蛋白水解酶也将通过酶谱图进行评估。 具体目的2：分离干扰弹力纤维形成的血清因子(S)。 PXE血清因子(S)的性质尚不清楚。我们建议确定PXE血清活性成分(S)的分子特征，并建立基于层析分离和质谱分析的分离方案。这些组分将使用体外功能分析进行测试。

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.