Mechanisms of intermittent parathyroid hormone effects on calcific aortic disease

 描述（由申请人提供）：钙化性主动脉血管和瓣膜疾病（CAVD）具有高死亡率，但没有可用的药物治疗。一个令人兴奋的可能性是，目前用于骨质疏松症的间歇性甲状旁腺激素（iPTH）治疗可能为CAVD提供治疗潜力，因为骨形成和CAVD之间存在与年龄无关的反比关系。事实上，在一项动物研究中，发现iPTH可预防CAVD的发生。然而，iPTH对既存CAVD受试者的影响仍不清楚。由于CAVD在需要iPTH治疗的人群中普遍存在，因此确定iPTH是否对既存CAVD患者仍然有益甚至有害至关重要。值得注意的是，与促进骨丢失和CAVD的持续高血清PTH水平相反，通过每日注射PTH产生的间歇性高水平促进骨骼骨形成。最近的发展表明，iPTH和氧化应激（CAVD的已知贡献者）之间存在意外联系。其他人在成骨细胞中的研究和我们在血管平滑肌细胞（VSMC）中的初步发现表明，iPTH降低了细胞氧化应激，表明了直接的受体介导的机制。我们最近的报告还提出了第二种机制，即在高脂血症小鼠中，iPTH通过诱导血清对氧磷酶-1（PON 1）活性降低促炎脂质氧化产物（氧化脂质）的循环水平。基于这些发现和我们先前的工作表明氧化脂质促进CAVD，我们假设iPTH将减弱预先存在的CAVD，并且将部分通过直接降低细胞氧化应激水平和部分通过经由PON 1间接降低氧化脂质的循环水平来实现。我们提出三个目标。在目标1中，将测试iPTH对高血脂小鼠中预先存在的CAVD的影响。由于钙化斑块含有iPTH作用的两个潜在靶点，即VSMC和前骨巨噬细胞，iPTH可能通过抑制骨软骨形成分化和/或诱导矿物质吸收来减弱CAVD。我们将评估：1）通过体内18 F <$microPET-microCT成像进行主动脉钙沉积，其允许对个体小鼠进行连续扫描，以及2）通过调节剂和标记物的水平进行氧化应激和骨软骨形成和骨细胞分化。在目标2中，将确定iPTH的作用位点。将使用PON 1缺陷型小鼠（Pon 1-/-ApoE-/-）评估全身机制，并使用血管缺陷型PTH受体1小鼠（Pth 1 r ΔVSMCApoE-/-）评估直接机制。在目标3中，我们将采用一种新的移植技术，其中将携带突变的1°鼠VSMC的扩散室皮下植入宿主小鼠中，我们将测试：1）iPTH与内源性PTH相关肽（PTHrP）对钙化的贡献和2）iPTH在糖尿病低骨转换骨质疏松症背景下的作用。研究结果将揭示iPTH是否是既存心血管疾病患者的CAVD治疗或风险，以及iPTH信号传导如何实现血管特异性效应。

 DESCRIPTION (provided by applicant): Calcific aortic vascular and valvular diseases (CAVD) carry a high mortality, yet no medical treatment is available. An exciting possibility is that intermittent parathyroid hormone (iPTH) treatment, currently used for osteoporosis, may offer therapeutic potential for CAVD, given that there is an age-independent, inverse relationship between bone formation and CAVD. Indeed, in an animal study, iPTH was found to prevent initiation of CAVD. However, the effect of iPTH in subjects with pre-existing CAVD is still not clear. Since CAVD is widespread and especially prevalent in the population requiring iPTH treatment, it is crucial to determine whether iPTH is still beneficial or even harmful in those wit pre-existing CAVD. Of note, in contrast to continuously high serum PTH levels, which promote bone loss and CAVD, intermittently high levels produced by daily injection of PTH, promote skeletal bone formation. Recent developments suggest an unexpected link between iPTH and oxidant stress, a known contributor of CAVD. Studies by others in osteoblasts and our preliminary findings in vascular smooth muscle cells (VSMC) show that iPTH reduced cellular oxidant stress, suggesting a direct receptor-mediated mechanism. A second mechanism is also suggested by our recent report showing that, in hyperlipidemic mice, iPTH reduces circulating levels of proinflammatory lipid oxidation products (oxylipids) by inducing serum paraoxonase-1 (PON1) activity. Based on these findings and our previous work showing that oxylipids promote CAVD, we hypothesize that, iPTH will attenuate pre-existing CAVD and will do so, in part, directly by reducing levels of cellular oxidant stress and, in part, indirectly by reducing circulaing levels of oxylipids via PON1. We propose 3 Aims. In Aim 1, iPTH effects will be tested on pre-existing CAVD in hyperlipidemic mice. Since calcific plaque contains two potential targets of iPTH action, VSMC and preosteoclastic macrophages, iPTH may attenuate CAVD by inhibiting osteochondrogenic differentiation and/or by inducing mineral resorption. We will assess: 1) aortic calcium deposition by in vivo 18F¯ microPET-microCT imaging, which allows serial scanning of individual mice and 2) oxidant stress and osteochondrogenic and osteoclastic differentiation by levels of regulators and markers. In Aim 2, the iPTH sites of action will be determined. The systemic mechanism will be assessed using PON1- deficient mice (Pon1-/-ApoE-/-), and the direct mechanism will be assessed using vascular deficient PTH receptor 1 mice (Pth1rΔVSMCApoE-/-). In Aim 3, we will employ a novel transplant technique, in which diffusion chambers carrying mutated 1°murine VSMC will be implanted subcutaneously into host mice, and we will test: 1) contributions of iPTH vs. endogenous PTH-related peptide (PTHrP) on calcification and 2) effects of iPTH in the context of low bone turnover osteoporosis of diabetes. The findings will reveal whether iPTH is a treatment or a risk for CAVD in patients with pre-existing cardiovascular disease, and how iPTH signaling achieves vascular-specific effects.