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.

概括主动脉瘤和解剖是Marfan综合征（MFS），最严重和致命的表现。预防主动脉词典的当前疗法仅适度有效。 MFS是由突变引起的或纤维蛋白-1（FBN1）中的缺失，这是细胞外微纤维的组成部分，周围并连接弹性纤维在主动脉介质中平滑肌细胞（SMC）。降低的纤维蛋白功能改变了信号细胞外基质和SMC，导致SMC凋亡和细胞外基质降解。另外，信号变化会导致活性氧（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以及激活蛋白激酶G（PKG）。尽管ROS增加，反应性氮（RN）和PKG激活有助于动脉瘤在MF中形成，ROS/RN的来源也不是氧化/亚硝化应激对SMC的影响功能已完全理解。维生素B12模拟核酰胺（我们持有多项专利）是强大的和多功能抗氧化剂，可以中和ROS和RN，包括NO。在最后一个赠款期间，我们表明具有激活PKG1突变（PRKG1R177Q）的小鼠会导致胸部动脉瘤和解剖在人类中，发展与增加的氧化应激和培养基变性酶的发展主动脉词。纤维碎裂，基质金属蛋白酶活性增加，培养基纤维化和SMC凋亡； Cobinamide 治疗完全阻止了这些变化。初步数据表明，Cobinamide还减少了主动脉扩张并防止MFS小鼠模型（FBN1C1041G/+）中的弹性纤维碎片和SMC凋亡，同时还原氧化应激和过量PKG信号的标记。我们假设增加了ROS/RN 结合不增加合成酶（NOS2）驱动异常SMC功能和主动脉的PKG激活 MFS中的病理学，以及优化的cobinamide剂量时间表将预防主动脉词和改善与MFS的小鼠中的生存，尤其是与阻滞剂结合使用时。在AIM 1中，我们将确定机甲 - 人纤维蛋白1缺陷或突变体中过量ROS/RNS产生的nism和后果（IPSC衍生） SMC，使用shRNA敲低和药物方法来抑制ROS/RNS生成酶和 PKG体外。我们将评估多余的无合酶和PKG活性对主动脉进展的贡献通过FBN1C1041G/+小鼠诱导的NOS2或PKG1诱导的SMC特异性基因敲除，体内疾病。我们还将测试通过SMC特异性过氧化氢酶过表达通过SMC特异性过表达的ROS解毒是否可以改善主动脉病理。在AIM2中，我们将确定最佳的核核苷剂量和开始时间以防止主动脉词和主动脉死亡中度（FBN1C1041G/+）和严重（FBN1MGR/MGR）MF的小鼠的解剖。此外，我们将将核酰胺与β受体阻滞剂普萘洛尔相结合，因为核酰胺可防止主动脉培养基变性，普萘洛尔降低了机械应力，而不会影响培养基的退化变化。这些研究在MFS患者中，可能导致对主动脉疾病的治疗可大大改善。