A novel treatment of aortic disease in Marfan Syndrome targeting oxidative stress and PKG dysregulation

针对氧化应激和 PKG 失调的马凡综合征主动脉疾病的新疗法

基本信息

批准号：
10453951
负责人：
RENATE B PILZ
金额：
$ 73.16万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10453951
关键词：
Acute Adhesions Adrenergic Agents Adrenergic Antagonists Adrenergic alpha-Antagonists Affect Age Alleles Aneurysm Antioxidants Aorta Aortic Aneurysm Aortic Diseases Aortic Rupture Apoptosis Attenuated Caliber Cardiovascular Abnormalities Cell physiology Cells Cessation of life Chest Collagen Complement Cyclic GMP Cyclic GMP-Dependent Protein Kinases DNA Data Deposition Development Disease Dissection Dose Drug Metabolic Detoxication Elastic Fiber Elastin Enzymes Extracellular Matrix Extracellular Matrix Degradation Extracellular Matrix Proteins Eye Abnormalities FBN1 Fibrosis Functional disorder Gene Expression Gene Expression Profile Generations Genetic Diseases Goals Grant Histology Human Human Milk Impairment In Vitro Inherited Knock-out Lead Legal patent Life Lipids LoxP-flanked allele Maintenance Marfan Syndrome Matrix Metalloproteinases Mechanical Stress Medial Microfibrils Mitochondria Mus Mutation Nitric Oxide Nitric Oxide Synthase Oral Oxidative Stress Pathology Pathway interactions Patients Peroxonitrite Persons Pharmacology Phenotype Play Prevalence Production Propranolol Protein Isoforms Proteins Reactive Nitrogen Species Reactive Oxygen Species Reporting Role Schedule Signal Transduction Smooth Muscle Myocytes Source Superoxide Dismutase Superoxides Tamoxifen Testing Thoracic Aortic Aneurysm Time Transgenes Up-Regulation Vitamin B 12 age related analog ascending aorta base catalase cobinamide drinking water extracellular fibrillin gain of function mutation improved in vivo induced pluripotent stem cell inhibitor knock-down loss of function mutation mimetics mouse model mutant nitrosative stress novel overexpression oxidation postnatal premature prevent public health relevance repaired skeletal abnormality small hairpin RNA stress kinase treatment strategy

项目摘要

Summary Aortic aneurysms and dissections are the most serious and deadly manifestations of Marfan Syndrome (MFS), and current therapies to prevent aortic dilation are only moderately effective. MFS is caused by mutations or deletions in fibrillin-1 (Fbn1), a component of extracellular microfibrils, which surround and connect elastic fibers to smooth muscle cells (SMCs) in the aortic media. Reduced fibrillin function alters signaling between extracellular matrix and SMCs, resulting in SMC apopotosis and extracellular matrix degradation. In addition, the altered signaling leads to increased production of reactive oxygen species (ROS) and nitric oxide (NO), increasing oxidative and nitrosative stress and activating protein kinase G (PKG) via the NO/cGMP pathway. Although the increased ROS, reactive nitrogen species (RNS), and PKG activation contribute to aneurysm formation in MFS, neither the sources of ROS/RNS nor the effects of oxidative/nitrosative stress on SMC functions are fully understood. The vitamin B12 analog cobinamide, on which we hold several patents, is a strong and versatile antioxidant that can neutralize ROS and RNS, including NO. During the last grant period, we showed that mice with an activating PKG1 mutation (Prkg1R177Q) that causes thoracic aneurysms and dissections in humans, develop aortic dilation associated with increased oxidative stress and media degeneration⸺elastic fiber fragmentation, increased matrix metalloproteinase activity, media fibrosis, and SMC apoptosis; cobinamide treatment completely prevented these changes. Preliminary data show that cobinamide also reduces aortic dilation and prevents elastic fiber fragmentation and SMC apoptosis in a mouse model of MFS (Fbn1C1041G/+), while reducing markers of oxidative stress and excess PKG signaling. We hypothesize that increased ROS/RNS combined with PKG activation from increased NO synthase (NOS2) drive abnormal SMC functions and aortic pathology in MFS, and that an optimized dose schedule of cobinamide will prevent aortic dilation and improve survival in mice with MFS, especially when combined with a -blocker. In Aim 1, we will determine the mecha- nisms and consequences of excess ROS/RNS generation in human fibrillin1-deficient or mutant (iPSC-derived) SMCs, using shRNA knockdown and pharmacological approaches to inhibit ROS/RNS-generating enzymes and PKG in vitro. We will assess contributions of excess NO synthase and PKG activity to the progression of aortic disease in vivo, by inducing SMC-specific knockout of NOS2 or PKG1 in Fbn1C1041G/+ mice. We will also test whether ROS detoxification by SMC-specific catalase overexpression ameliorates aortic pathology. In Aim2, we will determine the optimal cobinamide dose and starting time to prevent aortic dilation and death from aortic dissections in mice with moderate (Fbn1C1041G/+) and severe (Fbn1mgR/mgR) MFS, respectively. In addition, we will combine cobinamide with the β-blocker propranolol, because cobinamide prevents aortic media degeneration, while propranolol reduces mechanical stress without affecting degenerative changes in the media. These studies could lead to considerably improved treatment of the aortic disease in patients with MFS.

概括主动脉瘤和夹层是马凡综合征（MFS）最严重和致命的表现，目前预防主动脉扩张的疗法仅具有中等效果，是由突变或突变引起的。原纤维蛋白-1 (Fbn1) 缺失，原纤维蛋白是细胞外微原纤维的组成部分，围绕并连接弹性纤维主动脉中层平滑肌细胞 (SMC) 的原纤维功能降低会改变之间的信号传导。细胞外基质和 SMC，导致 SMC 凋亡和细胞外基质降解。信号传导的改变导致活性氧 (ROS) 和一氧化氮 (NO) 的产生增加，通过 NO/cGMP 途径增加氧化和亚硝化应激并激活蛋白激酶 G (PKG)。尽管 ROS、活性氮 (RNS) 和 PKG 激活增加会导致动脉瘤 MFS 中的形成，既不是 ROS/RNS 的来源，也不是氧化/亚硝化应激对 SMC 的影响我们拥有多项专利的维生素 B12 类似物椰酰胺的功能已得到充分了解。和多功能抗氧化剂，可以中和 ROS 和 RNS，包括 NO。研究表明，具有激活 PKG1 突变 (Prkg1R177Q) 的小鼠会导致胸动脉瘤和夹层在人类中，主动脉扩张与氧化应激增加和中膜变性⸺弹性相关纤维断裂、基质金属蛋白酶活性增加、介质纤维化和 SMC 凋亡；治疗完全阻止了这些变化。初步数据表明，cobinamide 还可以减少主动脉收缩。在 MFS (Fbn1C1041G/+) 小鼠模型中扩张并防止弹性纤维断裂和 SMC 凋亡，同时减少氧化应激标志物和过量 PKG 信号传导，我们捕获了 ROS/RNS 的增加。结合 NO 合酶 (NOS2) 增加引起的 PKG 激活，驱动异常 SMC 功能和主动脉 MFS 的病理学，并且 Cobinamide 的优化剂量方案将防止主动脉扩张并改善患有 MFS 的小鼠的存活率，特别是与 β 阻滞剂联合使用时。在目标 1 中，我们将确定机制。人原纤维蛋白 1 缺陷或突变体（iPSC 衍生）中 ROS/RNS 生成过多的现象和后果 SMC，使用 shRNA 敲除和药理学方法来抑制 ROS/RNS 生成酶和体外 PKG 我们将评估过量的 NO 合酶和 PKG 活性对主动脉进展的贡献。通过在 Fbn1C1041G/+ 小鼠中诱导 SMC 特异性敲除 NOS2 或 PKG1，我们还将测试体内疾病。 SMC 特异性过氧化氢酶过度表达的 ROS 解毒是否可以改善主动脉病理学。将确定最佳可宾酰胺剂量和开始时间，以防止主动脉扩张和主动脉死亡此外，我们还将分别对具有中度（Fbn1C1041G/+）和重度（Fbn1mgR/mgR）MFS的小鼠进行解剖。将可宾酰胺与 β 受体阻滞剂普萘洛尔联合使用，因为可宾酰胺可防止主动脉中膜变性，而普萘洛可降低机械应力而不影响介质的退行性变化。可能会显着改善 MFS 患者主动脉疾病的治疗。