Dynamic tissue-specific modulation of redox stress using chemogenetics

利用化学遗传学对氧化还原应激进行动态组织特异性调节

基本信息

批准号：
10393690
负责人：
Thomas Michel
金额：
$ 51.35万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-15 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10393690
关键词：
Abdomen Affect Age Aging Alanine Amino Acids Aneurysm Angiotensin II Receptor Animal Model Animals Aorta Aortic Aneurysm Biological Models Blood Vessels Breeding CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiovascular system Cells Code Country D-Amino Acid Dehydrogenase Dependovirus Development Diabetes Mellitus Disease Disease model Endothelial Cells Enterobacteria phage P1 Cre recombinase Epidemic Functional disorder Gene Expression Profile Generations Genetic Goals Heart Heart failure High Prevalence Human Hydrogen Peroxide LoxP-flanked allele MYH11 gene Mammalian Cell Measures Medical Metabolic syndrome Methodology Methods Modeling Monitor Morbidity - disease rate Mus Neurodegenerative Disorders Obesity Oxidation-Reduction Oxidative Stress Pathogenesis Pathologic Performance Pharmaceutical Preparations Pharmacology Pharmacotherapy Phenotype Physiological Play Population Prevalence Prevention Public Health Rattus Reactive Oxygen Species Recombinant Proteins Recombinants Research Scheme Science Site Smooth Muscle Myocytes Stress Terminator Codon Time Tissues Transgenes Transgenic Founder Mouse Transgenic Mice Transgenic Model Transgenic Organisms Validation Vascular Endothelial Cell Vascular Smooth Muscle Viral Vector Virus Yeasts base burden of illness cadherin 5 cost effective design disease phenotype experimental study feeding human disease in vivo mortality mouse model new therapeutic target novel novel therapeutic intervention novel therapeutics prevent programs promoter protein expression response single-cell RNA sequencing transcriptomics transgene expression ultrasound valsartan

项目摘要

This R33 research program proposes to characterize and validate novel transgenic mouse lines that exploit chemogenetics to create powerful models that will be used to identify new targets for the prevention and treatment of heart failure and aortic aneurysms. Most current animal models of heart failure and aortic aneurysm formation are limited by methodological complexities, and many models have limited relevance to human disease pathophysiology. Nearly all human heart failure and most arterial disease states are associated with high levels of reactive oxygen species (ROS) in affected tissues. We therefore developed a novel chemogenetic approach that dynamically modulates redox stress in vivo by exploiting a yeast D-amino acid oxidase (DAAO) that generates the ROS hydrogen peroxide (H2O2). DAAO is activated by D-amino acids, but not by the L-amino acids found in mammalian cells. We infected mice or rats with a recombinant cardiotropic adeno-associated virus isotype 9 (AAV9) that expresses DAAO. When animals are provided with D-amino acids, DAAO generates H2O2 in the heart, and the animals rapidly develop heart failure– which can be reversed by drug treatments. Although this approach is informative, a major limitation is the need to infect animals one at a time with the virus. Moreover, virus-based methods are constrained by the limited tissue selectivity of most viral vectors in vivo. Therefore, we generated new transgenic mouse lines designed to express DAAO in selected cardiovascular tissues. We have developed rigorous Performance Measures to quantitatively assess our progress in the proposed studies of these new transgenic lines. Aim 1 proposes experiments studying three independent transgenic founder lines expressing DAAO in the heart (driven by the MYH6 promoter) that will create a robust and cost-effective model to identify new pharmacological targets and new drugs to prevent and/or treat heart failure. In Aim 2, we propose to expand this approach to study redox stress in the vascular wall. We recently produced two new transgenic founder mouse lines containing DAAO in which a stop codon flanked by LoxP sites was cloned into the coding region of DAAO. By breeding these “floxed” DAAO transgenic lines with selected commercially-available lines expressing Cre recombinase under control of tissue-specific promoters, we will be able to dynamically regulate redox stress in different mouse tissues, permitting the study of the broad range of disease states in which redox stress is associated with pathogenesis. Here we will focus on DAAO expression in vascular endothelial cells (using VE-cadherin-Cre) and vascular smooth muscle cells (using MYH11-Cre) in order to identify the mechanisms whereby oxidative stress causes aortic aneurysms and to identify new drug targets to prevent or treat aneurysms. In both heart and aorta, we will perform state-of-the-art single cell RNA sequencing in order to track the changes in cell populations and transcriptional profiles that accompany development and regression of redox stress. The proposed studies will lead to the generation of powerful new model systems that will lead to the identification of novel targets and new drugs for the treatment and prevention of heart failure and aortic aneurysms.

这项R33研究计划的提案，以表征和验证利用新型的转基因小鼠系化学遗传学创建强大模型，该模型将用于确定预防的新目标治疗心力衰竭和主动脉瘤。大多数心力衰竭和主动脉瘤的动物模型形成受方法上的复杂性的限制，许多模型与人类疾病的相关性有限病理生理学。几乎所有人类心力衰竭和大多数动脉疾病状态都与高有关受影响组织中活性氧（ROS）的水平。因此，我们开发了一种新颖的化学生成通过利用酵母D-氨基酸氧化酶（DAAO）动态调节体内氧化还原应激的方法产生过氧化ROS氢（H2O2）。 Daao被D-氨基酸激活，但不会被L-Amino激活在哺乳动物细胞中发现的酸。我们用重组心脏腺相关病毒感染小鼠或大鼠表达DAAO的同型9（AAV9）。当动物配以D-氨基酸时，DAAO会产生H2O2 在心脏中，动物迅速发展为心力衰竭 - 可以通过药物治疗来逆转。虽然这种方法是有益的，一个主要的限制是需要一次感染一种病毒。而且，基于病毒的方法受体内大多数病毒载体的组织选择性有限。因此，我们生成了新的转基因小鼠系，旨在在选定的心血管组织中表达DAAO。我们有制定了严格的绩效指标，以定量评估我们在提议的研究中的进展这些新的转基因线。 AIM 1提案实验研究了三个独立的转基因创始人线在心脏（由MYH6启动子驱动）中表达Daao，这将创建一个强大而经济有效的模型确定新的药物靶标和新药物，以预防和/或治疗心力衰竭。在AIM 2中，我们建议扩展这种方法以研究血管壁中的氧化还原应激。我们最近产生了两个新的转基因创始人的鼠标线包含DAAO，其中将终止密码子侧翼的LOXP位点克隆到编码中 Daao地区。通过育种这些“ flox”的DAAO转基因线，并用选定的商业可用线在组织特异性启动子控制下表达CRE重组酶，我们将能够动态调节不同小鼠组织中的氧化还原应激，允许研究氧化还原的广泛疾病状态应力与发病机理有关。在这里，我们将重点介绍血管内皮细胞中的DAAO表达（使用VE-Cadherin-Cre）和血管平滑肌细胞（使用MyH11-CRE），以识别氧化应激会引起主动脉瘤并识别用于预防或治疗动脉瘤。在心脏和主动脉中，我们都将执行最新的单细胞RNA测序，以追踪细胞群体的变化和转录曲线，可满足氧化还原应激的发展和回归。拟议的研究将导致强大的新模型系统的产生，这将导致识别用于治疗和预防心力衰竭和主动脉瘤的新靶标和新药物的新药物。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Chemogenetic Approaches to Probe Redox Pathways: Implications for Cardiovascular Pharmacology and Toxicology.

DOI：
10.1146/annurev-pharmtox-012221-082339
发表时间：
2022-01-06
期刊：
Annual review of pharmacology and toxicology
影响因子：
12.5
作者：
通讯作者：

Differential endothelial hydrogen peroxide signaling via Nox isoforms: Critical roles for Rac1 and modulation by statins.

DOI：
10.1016/j.redox.2022.102539
发表时间：
2022-12
期刊：
REDOX BIOLOGY
影响因子：
11.4
作者：
Waldeck-Weiermair, Markus;Yadav, Shambhu;Kaynert, Jonas;Thulabandu, Venkata Revanth;Pandey, Arvind K.;Spyropoulos, Fotios;Covington, Taylor;Das, Apabrita Ayan;Kruger, Christina;Michel, Thomas
通讯作者：
Michel, Thomas

Redox à la carte: Novel chemogenetic models of heart failure.

氧化还原点菜：心力衰竭的新型化学遗传学模型。

DOI：
10.1111/bph.15093
发表时间：
2020
期刊：
British journal of pharmacology
影响因子：
7.3
作者：
Sorrentino,Andrea;Michel,Thomas
通讯作者：
Michel,Thomas

Sensory ataxia and cardiac hypertrophy caused by neurovascular oxidative stress in chemogenetic transgenic mouse lines.

DOI：
10.1038/s41467-023-38961-0
发表时间：
2023-05-29
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Yadav, Shambhu;Waldeck-Weiermair, Markus;Spyropoulos, Fotios;Bronson, Roderick;Pandey, Arvind K.;Das, Apabrita Ayan;Sisti, Alexander C.;Covington, Taylor A.;Thulabandu, Venkata;Caplan, Shari;Chutkow, William;Steinhorn, Benjamin;Michel, Thomas
通讯作者：
Michel, Thomas

Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting.

DOI：
10.1016/j.freeradbiomed.2021.10.023
发表时间：
2021-12
期刊：
Free radical biology & medicine
影响因子：
7.4
作者：
Erdogan YC;Altun HY;Secilmis M;Ata BN;Sevimli G;Cokluk Z;Zaki AG;Sezen S;Akgul Caglar T;Sevgen İ;Steinhorn B;Ai H;Öztürk G;Belousov VV;Michel T;Eroglu E
通讯作者：
Eroglu E