Role of Inhibitory SMADs in Calcific Aortic Valve Disease

描述(由申请人提供)： 抑制性Smads在钙性主动脉瓣病中的作用钙性主动脉瓣病(CAVD)在流行病学上与高脂血症有关，其生物学效应似乎通过氧化脂质、细胞因子和氧化应激之间的炎性正反馈环路发生。虽然这是一种迅速致命的疾病，但治疗选择极其有限。其临床严重程度是由于瓣叶僵硬，这限制了瓣膜的开启，增加了流出阻力。 和氧气需求，同时损害心脏灌流和氧气供应。因此，更好地了解钙化和硬化的潜在机制对于早期诊断和药物治疗的发展至关重要。CAVD被越来越多地认为是一个受调控的过程，涉及瓣膜细胞的成骨软骨分化。现在的证据表明，转化生长因子超家族成员的下游靶点，被称为“抑制小母亲对十足瘫痪”(I-SMADS；SMAD6和7)，调节瓣膜胚胎的发生，并可能作为CAVD的缓解因子。它们的上游效应分子BMP-2和转化生长因子-2在CAVD中均有表达，两者均可诱导瓣膜细胞钙化。它们通过受体相关的Smads(R-Smads)信号诱导骨软骨生成因子。尽管I-Smads最初被认为是负调控因子，但在某些情况下，它们已被证明促进转化生长因子超家族信号转导。此外，BMP-2和转化生长因子-2能够诱导瓣膜间质细胞聚集和收缩形成结节，类似于CAVD时瓣叶上的细胞，因此也可能促进叶硬化。在前期研究中，我们发现1)高脂血症小鼠主动脉瓣膜I-SMAD表达上调，2)肿瘤坏死因子诱导血管平滑肌细胞I-SMAD表达，3)蛋白激酶A(PKA)激活诱导I-SMAD表达并抑制体外培养的细胞结节形成。我们假设I-SMAD是由促炎因子诱导的，它们通过负向调节转化生长因子超家族诱导的CAVD来减轻CAVD。为了检验这一新的假设，我们提出了三个具体目标。在目标1中，我们将建立促炎因子在CAVD诱导I-SMAD中的作用机制。我们将使用体外(小鼠心脏瓣膜间质细胞；MVIC)、体外(小鼠心脏瓣膜器官培养；MVoC)和 在体内(PontgLdlr-/-Apob100和p75Tnfr-/-Ldlr-/-Apob100小鼠)方法。在目标2中，我们将测试I-Smads是否调节促炎症因子对CAVD的反应。我们将使用Smad6-/-小鼠及其缺乏I-SMAD的MVIC和MVoC，采用体外、体外和体内方法。氧化应激和炎症在体外是由氧化脂质和肿瘤坏死因子引起的，在体内是由腺嘌呤饮食引起的。在目标3中，我们将测试I-Smads是否介导了PKA对瓣膜结节形成和收缩的抑制作用。我们将使用胶原-凝胶收缩和结节形成试验以及I-SMAD基因沉默。拟议工作的结果将利用I-SMADS的管理能力来制定预防措施和治疗CAVD。 (摘要结束)

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- (TGF-) 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-, 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- superfamily signaling. In addition, BMP-2 and TGF- 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- 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. (End Abstract)