Impact of microfibril turnover on vascular development and disease

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.