ISOLATION OF THE SERUM FACTOR RESPONSIBLE FOR PSEUDOXANTHOMA ELASTICUM

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目标.这项研究的长期目标是更好地了解弹性纤维的显着生物学和结构组织。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。我们的项目的具体目标是确定和表征的分子事件，导致弹性假黄瘤（PXE）的弹性纤维的改变和钙化作为一个相关的疾病模型，应该有助于阐明动态管理的弹性纤维蛋白的稳态。 具体目的1：确定体外弹性纤维在PXE血清作用下的组装缺陷。 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，培养基中补充有来自PXE患者或正常个体的血清。将通过免疫组织学检查主要弹性纤维组分（弹性蛋白、纤蛋白-4、-5、纤蛋白-1、-2、MAGP 1、LOX和LOXL），并在弹性纤维沉积的各个阶段（从汇合前到汇合后）进行定量。将通过电子显微镜评价结构变化。还将通过酶谱法评价在存在PXE血清的情况下成纤维细胞和平滑肌细胞产生弹性纤维特异性蛋白酶的情况。 具体目标2：分离干扰弹性纤维形成的血清因子。 PXE血清因子的性质尚不清楚。我们建议确定PXE血清活性成分的分子特征，并建立一个分离方案的基础上色谱分离，然后进行质谱分析。将使用体外功能试验检测这些组分。

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.