Proteoglycan Regulation During Cardiac Valve Development and Homeostasis

描述(申请人提供)：成人心脏瓣膜疾病影响高达5%的美国人，大约25%的老年人口发展为主动脉瓣硬化症，这是瓣膜疾病的标志。瓣膜功能不全的主要治疗方法是手术干预，在美国，每年的费用估计为10亿美元。大多数患病的瓣膜都有潜在的畸形，这一事实表明，确定瓣膜疾病的病因取决于发现与瓣膜发育有关的新机制和基因。然而，在发现瓣膜疾病的根源方面进展有限，部分原因是缺乏显示心脏瓣膜表型显著外显性的可存活的小鼠模型。发育中的粘液瘤瓣膜异常和成人疾病的一个特征是细胞外基质(ECM)层积的丧失和ECM蛋白多糖的积聚，包括Verscan。ADAMTS(具有血栓反应蛋白1型基序的去整合素和金属蛋白酶结构域)蛋白多糖是一类特殊的细胞外基质金属蛋白酶，能裂解蛋白多糖。ADAMTS5缺乏的小鼠在妊娠后期会出现严重的心脏瓣膜增大，并显著积累ADAMTS5的ECM底物Verscan。在体内，Verscan的还原挽救了Adamts5/-瓣膜的表型，并证明了Verscan的切割是其从临时ECM清除所必需的。到目前为止，Verscan是唯一已知的ADAMTS5在ADAMTS5缺陷瓣膜中积累的ECM底物。来自Adamts5-/-小鼠的瓣膜前间充质细胞维持Sox9的表达，并显示出高水平的增殖，表明它们不像它们的WT对应物那样分化。我们发表的数据还表明，Verscan的ECM积聚抑制了Smad2的磷酸化。Adamts5-/-与Smad2-/-的异种杂交加剧了瓣膜的表型，并导致二尖瓣的高外显率(80%)。在这项应用中，我们试图阐明ECM-细胞信号机制，这些信号机制是响应蛋白多糖的裂解而激活的，并与瓣膜成熟和动态平衡整合在一起。由于ADAMTS5肽抑制剂正处于缓解骨关节炎的临床试验中，研究蛋白多糖在正常瓣膜发育和稳态中的切割需求具有直接的临床意义。我们应用的三个具体目的验证了我们的总体假设，即ADAMTS5介导的多功能切割通过与控制细胞分化和富含蛋白多糖的细胞外基质和纤维状细胞外基质的平衡的TGFbeta信号相互连接，在半月形心脏瓣膜成熟过程中发挥关键作用。目的1：探讨半月瓣发育过程中ADAMTS5依赖的ECM重塑上调Smad2磷酸化的机制。目的：确定ADAMTS5在半月瓣发育过程中重建临时心内膜细胞外基质所需的时空表达。目的：研究ADAMTS5缺乏对成年小鼠心脏瓣膜ECM及功能的影响。

DESCRIPTION (provided by applicant): Adult cardiac valve disease affects up to 5% of Americans with approximately 25% of the aged population developing aortic valve sclerosis, a marker of valve disease. The primary treatment of valvular insufficiency is surgical intervention with an estimated cost of $1 billion per year in the US. The fact that most diseased valves have an underlying malformation suggests that determining the etiology of valve diseases lies in the discovery of novel mechanisms and genes that are involved in valve development. However, progress in discovering the roots of valve disease has been limited in part due to the lack of viable mouse models that exhibit significant penetrance of a cardiac valve phenotype. A hallmark of both developmental myxomatous valve abnormalities and adult disease is the loss of stratification of extracellular matrix (ECM) and the accumulation of the ECM proteoglycans including versican. The ADAMTS (ADisintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) proteoglycanases are a specific class of extracellular matrix metalloproteases that cleave proteoglycans. Mice deficient in ADAMTS5 develop severely enlarged cardiac valves by late gestation with significant accumulation of versican, the ECM substrate of ADAMTS5. In vivo reduction of versican, rescues the Adamts5-/- valve phenotype, and demonstrates that cleavage of versican is required for its clearance from the provisional ECM. To date, versican is the only ECM substrate of ADAMTS5 known to accumulate in ADAMTS5 deficient valves. Prevalvular mesenchymal cells from the Adamts5-/- mice maintain Sox9 expression and show elevated levels of proliferation suggesting they do not differentiate like their WT counterparts. Our published data also show that ECM accumulation of versican inhibits Smad2 phosphorylation. Intergenetic cross of the Adamts5-/- with Smad2-/- exacerbates the valve phenotype and results in a high penetrance (80%) of bicuspid aortic and pulmonary valves. In this application we seek to elucidate ECM-cell signaling mechanisms that are activated in response to proteoglycan cleavage and integral to the valve maturation and homeostasis. Since ADAMTS5 peptide inhibitors are in clinical trials to alleviate osteoarthritis, investigation into the requirement of proteoglycan cleavage in normal valve development and homeostasis has immediate clinical relevance. The three specific aims of our application test our overarching hypothesis that ADAMTS5 mediated versican cleavage plays a pivotal role in semilunar cardiac valve maturation through interconnections with TGFbeta signaling that control cell differentiation and the balance of proteoglycan-rich and fibrous ECM. Aim 1: Determine the mechanism by which ADAMTS5 dependent ECM remodeling upregulates Smad2 phosphorylation during semilunar valve development. Aim 2: Determine the spatiotemporal expression of ADAMTS5 that is required to remodel the provisional ECM of endocardial cushions during semilunar valve development. Aim 3: Determine the consequence of ADAMTS5 deficiency on adult murine valve ECM and function.