Impact of microfibril turnover on vascular development and disease

摘要 显性FBN1突变导致马凡综合征，这是一种遗传性人类结缔组织疾病，影响 纤维蛋白-1微纤维，并导致胸主动脉瘤，并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，它在机械中提供了重要的纽带 从SMC收缩细胞骨架到细胞外基质的连续体，除了提供模板 用于弹性纤维组装。FBN1单倍体功能不全导致组织纤维蛋白-1含量降低被认为是 是相当大比例(超过1/3)马凡综合征突变的机制。 在最近的工作中，我们发现VSMC分泌的一种金属蛋白酶ADAMTS6可以同时裂解纤维蛋白-1和 纤维蛋白-2。后者主要在胚胎期产生，被认为在 出生后的大动脉。对不能在出生后存活的小鼠Adamts6零突变体的分析表明， 纤维蛋白-1和纤维蛋白-2的堆积，并有遗传上的主要骨骼和心脏缺陷 归因于纤维蛋白-2的积聚。因此，ADAMTS6似乎是调节纤维蛋白微原纤维的主要蛋白酶。 营业额。这为将ADAMTS6靶向马凡综合征治疗一种新疾病提供了令人信服的理由- 修改方法。 基于这些发现，这一提议的首要假设是ADAMTS6在血管中失活 出生后的平滑肌细胞将保护主动脉原纤维-1微纤维不被蛋白水解性转换，从而增加 马凡综合征患者微纤维的丰富和主动脉瘤生长和夹层的减轻。在目标1中，我们 将使用一个新的Adamts6条件突变体来验证这一假设，方法是在 严重马凡综合征可靠进展为夹层的特征良好的小鼠模型中的VSMCs 和破裂。在目标2中，我们将定义纤维蛋白-1和ADAMTS6的分子间相互作用，以确定 蛋白质降解的主要分子决定因素。在体外，微纤维组装将被用来测试 阻断ADAMTS6-纤维素间的相互作用。这些实验将为未来的保护方法提供信息 ADAMTS6介导的周转过程中形成的微纤维。 影响：马凡综合征的疾病修正方法不存在，非手术选择也不存在 在预防夹层方面完全有效。这些目的充分利用了我们最初发现的ADAMTS6裂解 纤维蛋白-1用于持续研究，旨在推动一种基于ADAMTS6阻断的疾病的发展- 马凡综合征的改良手术入路。具体地说，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不被破坏来增加微纤维的丰度。同舟共济 这些目标为可能的疾病修正疗法(目标1)提供了原则性证明，并为干预提供了基础。 用ADAMTS6裂解纤维蛋白-1(目标2)。这里提出的工作也解决了基本问题 纤维蛋白-1是如何在血管壁上翻转的。

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.