Impact of microfibril turnover on vascular development and disease

总结 显性FBN 1突变导致马凡综合征，一种遗传性人类结缔组织疾病， Thrombin-1微纤维并导致胸主动脉瘤，具有主动脉夹层和破裂的风险。原纤维蛋白- 1是血管平滑肌细胞（VSMC）的产物，其提供了机械的重要环节， 从SMC收缩性细胞骨架到细胞外基质的连续体，除了提供模板外， 用于弹性纤维组装。由于FBN 1单倍不足而导致的组织中BFN-1含量的降低被认为 是马凡氏综合征突变的重要比例（超过1/3）的机制。 在最近的工作中，我们发现ADAMTS 6，一种由VSMC分泌的金属蛋白酶，既能切割β-内酰胺酶-1， 抗氧化蛋白-2后者主要在胚胎期产生，被认为在发育过程中起次要作用。 出生后的大动脉对小鼠Adamts 6无效突变体的分析显示， 积累的两个resistin-1和resistin-2，与主要的骨骼和心脏缺陷，我们有遗传 这归因于Escherichia coli-2积累。因此，ADAMTS 6似乎是微纤维中调节细胞凋亡的主要蛋白酶 周转这为靶向ADAMTS 6治疗马凡氏综合征这一新型疾病提供了令人信服的理论基础- 修改方法。 基于这些发现，该提议的总体假设是血管中ADAMTS 6失活是由血管内皮细胞中的ADAMTS 6失活引起的。 出生后平滑肌细胞将保护主动脉内皮素-1微纤维免于蛋白水解周转，从而增加 微纤维丰度和减轻马凡氏综合征的主动脉瘤生长和夹层。目标1： 将使用一种新的Adamts 6条件突变体，通过条件性缺失Adamts 6来验证这一假设， 严重马凡氏综合征小鼠模型中的VSMCs可靠地进展为夹层 和破裂在目的2中，我们将定义Escherin-1和ADAMTS 6的分子间相互作用，以鉴定Escherin-1和ADAMTS 6的相互作用。 蛋白水解的主要分子决定因素。将使用体外微原纤维组装来测试以下物质的影响： 阻断ADAMTS 6-β-内酰胺酶相互作用。这些实验将为未来的保护方法提供信息。 微纤维从ADAMTS 6介导的周转。 影响：马凡氏综合征的疾病修饰方法不存在，非手术选择也不存在。 完全有效地防止了解剖。这些目标利用了我们最初的发现，即ADAMTS 6切割 用于旨在推动ADAMTS 6阻断型疾病发展的持续研究- 马凡氏综合征的改良方法。具体而言，许多患者的疾病机制是减少 我们的目标是通过保护微纤维免受破坏来提高微纤维的丰度。一起 这些目标为可能的疾病改善疗法（目标1）提供了原理证明，并为干预提供了基础。 用ADAMTS 6切割人精子蛋白-1（Aim 2）。本文提出的工作还涉及一些基本问题， 在血管壁中是如何翻转的。

SUMMARY Dominant FBN1 mutations cause Marfan syndrome, an inherited human connective tissue disorder affecting fibrillin-1 microfibrils and leading to thoracic aortic aneurysms with risk of aortic dissection and rupture. Fibrillin- 1 is a product of vascular smooth muscle cells (VSMC), which provides an important link in the mechanical continuum from the SMC contractile cytoskeleton to the extracellular matrix, in addition to providing a template for elastic fiber assembly. Reduced tissue fibrillin-1 content as a result of FBN1 haploinsufficiency is thought to be the mechanism underlying a significant proportion (over 1/3) of Marfan syndrome mutations. In recent work we found that ADAMTS6, a metalloprotease secreted by VSMC, cleaves both fibrillin-1 and fibrillin-2. The latter is produced primarily during the embryonic period and is thought to have a minor role in the aorta after birth. Analysis of a mouse Adamts6 null mutant, which does not survive past birth, shows an accumulation of both fibrillin-1 and fibrillin-2, with major skeletal and cardiac defects we have genetically attributed to fibrillin-2 accumulation. Thus, ADAMTS6 appears to be a major protease regulating fibrillin microfibril turnover. This provides a compelling rationale for targeting ADAMTS6 in Marfan syndrome in a novel disease- modifying approach. Based on these findings, the overarching hypothesis of this proposal is that ADAMTS6 inactivation in vascular smooth muscle cells postnatally will protect aortic fibrillin-1 microfibrils from proteolytic turnover, thus increasing microfibril abundance and mitigating aortic aneurysm growth and dissection in Marfan syndrome. In Aim 1, we will use a new Adamts6 conditional mutant to test this hypothesis through conditional deletion of Adamts6 in VSMCs in a well-characterized mouse model of severe Marfan syndrome that reliably progresses to dissection and rupture. In Aim 2, we will define the intermolecular interaction of fibrillin-1 and ADAMTS6 to identify the major molecular determinants of proteolysis. In vitro microfibril assembly will be used to test the impact of blocking ADAMTS6-fibrillin interactions. These experiments will inform future approaches for protecting microfibrils from ADAMTS6-mediated turnover. Impact: A disease-modifying approach for Marfan syndrome does not exists, and non-surgical options have not been wholly effective in preventing dissection. These aims leverage our initial discovery that ADAMTS6 cleaves fibrillin-1 for continued investigations intended to drive development of an ADAMTS6 blockade-based disease- modifying approach for Marfan syndrome. Specifically, the disease mechanism in many patients is reduction of fibrillin-1 microfibrils and we aim to enhance microfibril abundance by protecting them from breakdown. Together the aims provide a proof of principle for a possible disease-modifying therapy (Aim 1) and the basis for interfering with ADAMTS6 cleavage of fibrillin-1 (Aim 2). The work proposed herein also addresses fundamental questions of how fibrillin-1 is turned over in the vascular wall.