Role of Inhibitory SMADs in Calcific Aortic Valve Disease

描述（由申请人提供）： 抑制性SMAD在钙化性主动脉瓣疾病中的作用钙化性主动脉瓣疾病（CAVD）在流行病学上与高脂血症相关，其生物学效应似乎通过连接氧化脂质、细胞因子和氧化应激的炎性正反馈回路发生。虽然它是一种快速致命的疾病，但治疗选择非常有限。其临床严重程度是由于瓣叶硬化，这限制了瓣膜打开，增加了流出阻力 和氧气需求，同时损害心脏灌注和氧气供应。因此，更好地了解钙化和硬化的机制对于早期诊断和药物治疗的发展至关重要。CAVD越来越多地被认为是一个受调控的过程，涉及瓣膜细胞的骨软骨分化。现在有证据表明，转化生长因子-β（TGF-β）超家族成员的下游靶点，称为“抑制性小母体对十肢瘫痪”（I-SMAD; SMAD 6和7），调节瓣膜胚胎发生，并可作为CAVD的缓解因子。它们的上游效应物BMP-2和TGF-β在CAVD中表达，并且都诱导瓣膜细胞钙化。它们通过受体相关SMADs（R-SMADs）信号传导以诱导骨软骨形成因子。虽然I-SMAD最初被鉴定为负调节因子，但在某些条件下，它们已被证明促进TGF-β超家族信号传导。此外，BMP-2和TGF-β还可能促进瓣叶硬化，因为它们能够诱导瓣膜间质细胞聚集和收缩成结节，类似于CAVD中瓣叶上的结节。在初步研究中，我们发现：1）高血压小鼠主动脉瓣中I-SMAD表达上调，2）TNF-α诱导主动脉平滑肌细胞中I-SMAD表达，3）蛋白激酶A（PKA）激活诱导I-SMAD表达并抑制体外细胞结节形成。我们假设I-SMAD是由促炎因子诱导的，并且它们通过负调节TGF-β超家族诱导的CAVD来减轻CAVD。为了验证这一新的假设，我们提出了三个具体目标。目的一：探讨促炎因子对CAVD诱导I-SMAD的作用机制。我们将使用体外（鼠瓣膜间质细胞; mVIC）、离体（鼠瓣膜器官培养物; mVOC）和体内（PontgLdlr-/-Apob 100和p75 Tnfr-/-Ldlr-/-Apob 100小鼠）方法。在目标2中，我们将测试I-SMADs是否响应促炎因子调节CAVD。我们将使用Smad 6-/-小鼠及其I-SMAD缺陷型mVIC和mVOC采用体外、离体和体内方法。氧化应激和炎症将在体外由氧化脂质和TNF-α诱导，在体内由腺嘌呤饮食诱导。在目的3中，我们将测试I-SMADs是否介导PKA对瓣膜结节形成和收缩的抑制作用。我们将使用I-SMAD基因沉默的胶原凝胶收缩和结节形成测定。拟议工作的结果将利用I-SMAD的监管权力，制定CAVD的预防措施和医疗方法。

DESCRIPTION (provided by applicant): Role of Inhibitory SMADs in Calcific Aortic Valve Disease Calcific aortic valve disease (CAVD) is associated epidemiologically with hyperlipidemia, the biological effects of which appear to occur through an inflammatory, positive feedback loop linking oxidized lipids, cytokines, and oxidant stress. Although it is a rapidly fatal disorder, therapeutic options are extremely limited. Its cliical severity is due to leaflet stiffening, which restricts valve opening, increasing outflow resistance and oxygen demand, while impairing cardiac perfusion and oxygen supply. Thus, a better understanding of the mechanisms underlying calcification and stiffening is critical for the development of early diagnosis and medical therapy. CAVD is increasingly acknowledged as a regulated process, involving osteochondrogenic differentiation of valvular cells. Evidence now suggests that the downstream targets of members of the transforming growth factor-ß (TGF-ß) superfamily, known as "inhibitory small mothers against decapentaplegic" (I-SMADs; SMAD6 and 7), regulate valve embryogenesis and may serve as a mitigating factor in CAVD. Their upstream effectors, BMP-2 and TGF-ß, are expressed in CAVD, and both induce valvular cell calcification. They signal through receptor-associated SMADs (R-SMADs) to induce osteochondrogenic factors. Although I-SMADs were originally identified as negative regulators, under some conditions, they have been shown to promote TGF-ß superfamily signaling. In addition, BMP-2 and TGF-ß may also promote leaflet stiffening, given their ability to induce aggregation and contraction of valvular interstitial cells into nodules resembling those on leaflet in CAVD. In preliminary studies, we found that 1) I-SMAD expression is upregulated in aortic valves in hyperlipidemic mice, 2) TNF- induces I-SMAD expression in aortic smooth muscle cells, and 3) protein kinase A (PKA) activation induces I-SMAD expression and inhibits cellular nodule formation in vitro. We hypothesize that I-SMADs are induced by pro-inflammatory factors and that they mitigate CAVD by negatively regulating TGF-ß superfamily-induced CAVD. To test this novel hypothesis, we propose three Specific Aims. In Aim 1, we will establish the mechanism of effects of pro-inflammatory factors on I-SMAD induction in CAVD. We will use in vitro (murine valvular interstitial cells; mVIC), ex vivo (murine valvular organ culture; mVOC) and in vivo (PontgLdlr-/-Apob100 and p75Tnfr-/-Ldlr-/-Apob100 mice) approaches. In Aim 2, we will test whether I-SMADs regulate CAVD in response to pro-inflammatory factors. We will employ in vitro, ex vivo, and in vivo approaches using Smad6-/- mice and their I-SMAD-deficient mVIC and mVOC. Oxidant stress and inflammation will be induced by oxidized lipids and TNF- in vitro and by an adenine diet in vivo. In Aim 3, we will test whether I-SMADs mediate inhibitory effects of PKA on formation and contraction of valvular nodules. We will use collagen-gel contraction and nodule formation assays with I- SMAD gene silencing. Results of the proposed work will harness the regulatory power of I-SMADs for development of preventive measures and medical treatments for CAVD.