Proteoglycan Metabolism During Cardiac Valve Development and Disease

摘要：大约1.5%的人有可能导致二尖瓣关闭不全的二尖瓣 血流和器官损害(1，2)。BAV也是升主动脉壁的独立危险因素 可能导致破裂和猝死的并发症。然而，BAV的细胞和分子基础是 未知。由于来自多个细胞系的贡献，即使是BAV形成的基本方面也是未知的 以及缺乏高渗透性的可行的BAV小鼠模型。功能不全的主动脉瓣显示出巨大的 细胞外基质(ECM)、蛋白多聚糖(Vcan)和聚集素(Acan)的积累(3-7) 蛋白多糖过量的来源和后果在很大程度上是未知的。由于蛋白多糖高度稳定， 丰度主要由蛋白降解来调节，这导致我们研究了蛋白多糖的作用。 乳沟。我们发现单个ECM蛋白葡聚糖酶Adamts5的缺失会导致主动脉瓣增大 伴升主动脉病变(100%)。这些缺陷与Vcan和Acan在 受影响的组织(8-10个)，反映人类的情况。机械上，成人5-/-主动脉瓣原基 在Vcan过多的情况下，pSmad2减少，并且当通过生成Adam进一步减少Smad2时，5-/- Smad2+/-小鼠，BAV的外显率较高(75%)，远高于其他小鼠BAV模型。 这项建议的目标是利用存活的Adamts5-/-；Smad2+/-小鼠，这些小鼠表现出高比例的 BAV，以定义当被干扰时有助于BAV形成的形态事件、细胞行为和因素。 由于Vcan过多是瓣膜功能障碍和病变的标志，因此使用具有影响 Vcan含量的控制，可能有助于阐明蛋白多糖代谢是如何在发育和 也可能让我们对疾病有更深入的了解。实验验证了ECM Vcan裂解坐标的假设 三尖瓣形态所需的间充质细胞行为和心肌细胞贡献 主动脉瓣。这一假说有两个目的：Aim 1测试改变的Vcan裂解对 主动脉瓣形成所需的间充质细胞谱系行为。我们的初步数据显示， 在发育过程中，Vcan裂解片段的丢失和多余的完整Vcan，扰乱了谱系特有的模式 BAV Adamts5-/-；Smad2+/-小鼠的主动脉瓣。体外和体内方法的结合将破译 依赖于Vcan裂解的谱系特异性细胞行为和指导因素，以及防止 BAV。尽管Dogma声称瓣膜尖端来自间充质细胞，但Aim 2研究了 Vcan过多对大鼠主动脉瓣发育过程中心肌细胞系的影响 Adamts5-/-；Smad2+/-小鼠和其他BAV小鼠模型。我们的初步数据显示心肌谱系 Adamts5的表达是清除富含Vcan的ECM和形成主动脉瓣非冠状瓣叶所必需的。 由于BAV中新出现的VCAN过剩流行，对ECM营业额的调查将使我们能够洞察 研究主动脉瓣疾病的分子和细胞起源，这可能导致治疗方面的进展。

Summary: Approximately 1.5% of individuals have a Bicuspid Aortic Valve (BAV) that can result in insufficient blood flow and organ damage (1, 2). A BAV is also an independent risk factor for ascending aortic wall complications that can lead to rupture and sudden death. However, the cell and molecular basis for the BAV is unknown. Even basic aspects of BAV formation are undefined due to contributions from multiple cell lineages and a lack of highly penetrant viable BAV mouse models. Dysfunctional aortic valves exhibit massive accumulation of the extracellular matrix (ECM) proteoglycans versican (Vcan) and aggrecan (Acan) (3-7) but the origin and consequence of excess proteoglycans is largely unknown. Since proteoglycans are highly stable, abundance is regulated primarily by proteolytic degradation which led us to investigate a role for proteoglycan cleavage. We discovered that loss of a single ECM proteoglycanase, Adamts5, results in enlarged aortic valves with ascending aortopathies (100%). These defects co-localize with substantial increases in Vcan and Acan in the affected tissues (8-10) that mirror the human condition. Mechanistically, in Adamts5-/- aortic valve primordia with excess Vcan there is a reduction of pSmad2, and when Smad2 is reduced further by generating Adamts5-/- ;Smad2+/- mice, there is a high penetrance of BAV (75%), much higher than seen in other mouse BAV models. The objective of this proposal is to utilize the viable Adamts5-/-;Smad2+/- mice that exhibit a high percentage of BAV, to define morphological events, cell behaviors and factors that when disrupted contribute to BAV formation. Since excess Vcan is a hallmark of dysfunctional and diseased valves, use of a model with mutations that impact the control of Vcan content, may shed light on how proteoglycan metabolism is regulated in development and may also give insight into disease. Experiments test the hypothesis that ECM Vcan cleavage coordinates mesenchymal cell behaviors and myocardial cell contributions that are required for the tricuspid morphology of aortic valves. The hypothesis is tested in two aims: Aim 1 tests the impact of altered Vcan cleavage on mesenchymal cell lineage behaviors that are required for aortic valve formation. Our preliminary data show that loss of Vcan cleaved fragments and excess intact Vcan, disrupted lineage-specific patterning in the developing aortic valves of the BAV Adamts5-/-;Smad2+/-mice. A combination of ex vivo, and in vivo approaches will decipher the lineage-specific cell behaviors, and guidance factors that are dependent on Vcan cleavage and to prevent BAV. Although dogma states that valve cusps arise from mesenchymal cells, Aim 2 investigates the consequence of excess Vcan on the myocardial cell lineage contributions to the developing aortic valve in Adamts5-/-;Smad2+/- mice and other murine models of BAV. Our preliminary data show myocardial lineage expression of Adamts5 is required to clear Vcan-rich ECM and to form the non-coronary cusp of the aortic valve. Due to the emerging prevalence of excess Vcan in BAV, the investigation into ECM turnover will allow insight into the molecular and cellular origins of aortic valve diseases, which may lead to therapeutic advances.