Development of RhoA Optical Sensor Mice for Novel Vascular Smooth Muscle Studies

开发用于新型血管平滑肌研究的 RhoA 光学传感器小鼠

基本信息

批准号：
8683411
负责人：
MEGAN A RIZZO
金额：
$ 19.19万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8683411
关键词：
Adrenergic Agents Angiotensin II Animals Area Arteries Binding Biology Biosensor Bladder Blood Vessels Cause of Death Cell Culture Techniques Cell Proliferation Cell Surface Receptors Cells Characteristics Development Diabetes Mellitus Diabetic mouse Discipline Disease Disease model Ear Endothelin Enzymes Figs - dietary Fluorescence Microscopy Fluorescence Resonance Energy Transfer Foundations G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Gene Transfer Techniques Guanine Nucleotide Exchange Factors Guanosine Triphosphate Health Heart Diseases Heart failure Hyperactive behavior Hypertension Image Inflammatory Investigation Life Light Measurement Measures Metabolic syndrome Methodology Methods Modeling Molecular Conformation Monomeric GTP-Binding Proteins Mus Muscle Contraction Myosin ATPase Nature Nerve Neuraxis Optics Pathogenesis Performance Phosphorylation Physiological Physiology Proteins Reagent Receptor Signaling Regulation Reporting Research Research Personnel Resolution Rho-associated kinase Role Signal Transduction Skin Smooth Muscle Smooth Muscle Myocytes Stroke Sympathetic Nervous System Testing Time Tissues Transgenic Mice Transgenic Organisms Translating United States Vascular Diseases Vascular Smooth Muscle Vascular remodeling Work adrenergic animal tissue arteriole base design femoral artery gastrointestinal human disease imaging modality in vivo in vivo imaging mature animal mouse model myosin phosphatase novel optical sensor promoter receptor response sensor two-photon vasoconstriction

项目摘要

DESCRIPTION (provided by applicant): Leading causes of death, such as heart disease, stroke, and diabetes, and are all associated with vascular dysfunction. Thus, understanding the physiologic mechanisms that control vascular function is vital for understanding the pathogenesis of these conditions and for developing new treatments. Many important classes of vasomodulators work by binding to G-protein coupled receptors (GPCRs) that initiate signaling cascades that converge on the small GTPase, RhoA. RhoA-GTP activates Rho-associated kinase (ROK), which regulates contraction of smooth muscle through inhibition of myosin light chain phosphatase (MLCP) and is also involved in pathophysiological responses of arteries; vascular remodeling, smooth muscle cell proliferation, and recruitment of inflammatory cells. RhoA can therefore be regarded as an integrative control point that translates diverse GPCR signaling to numerous artery functions. The fraction of RhoA molecules that are bound to GTP constitutes the 'fractional activation' of RhoA, and is a quantitative measure of the potential activation of ROK. In preliminary work we have constructed a high performance FRET-based RhoA activation sensor molecule, RhoA.v2. RhoA.v2 utilizes mCerulean3 and mCitrine to provide outstanding characteristics for quantitative FRET measurements, particularly with two-photon excitation. Two-photon excitation also provides the ability to image RhoA.v2 within cells of intact tissues of the living mouse and even entirely non-invasively, through the skin. The major Aims of this proposal are to 1) develop a novel transgenic mouse model that expresses RhoA.v2 specifically in smooth muscle cells, 2) develop methods, utilizing two- photon imaging, that unlock the full quantitative power inherent to the design of the RhoA.v2, such that the fractional activation of RhoA can be quantified in arteries in vivo, and 3) pursue a preliminary investigation into the role of RhoA in control of contraction of smooth muscle cells in arteries by the sympathetic nervous system (SNS) activity. SNS hyperactivity, which can exist only in living animals, is a key factor in hypertension, metabolic syndrome, heart failure and other conditions. We will test the hypothesis that RhoA is a critical effector of SNS in certain arteries in vivo. Ths work will be accomplished by a team of investigators with complimentary expertise in optical probe development/FRET imaging (Dr. Rizzo) and vascular biology and in vivo imaging (Dr. Wier). In summary, a novel RhoA biosensor mice will be created and methods, utilizing two-photon imaging, will be developed for quantification of RhoA activation in vivo, with subcellular resolution. The model and methods developed by this proposal will be broadly impactful to hypertension, diabetes, many areas of vascular biology (including stroke), and areas of general smooth muscle involvement, such as gastrointestinal and bladder function.

描述（由申请人提供）：死亡的主要原因，例如心脏病，中风和糖尿病，都与血管功能障碍有关。因此，了解控制血管功能的生理机制对于理解这些疾病的发病机理和开发新治疗至关重要。许多重要类的血管调节剂通过与G蛋白偶联受体（GPCR）结合来起作用，这些受体（GPCR）启动了在小GTPase RhoA上收敛的信号传导级联反应。 RhoA-GTP激活了Rho相关激酶（ROK），该激酶通过抑制肌球蛋白轻链磷酸酶（MLCP）来调节平滑肌的收缩，并且还参与了动脉的病理生理反应。血管重塑，平滑肌细胞增殖和炎症细胞的募集。因此，RhoA可以被视为一个综合控制点，将各种GPCR信号转化为众多动脉功能。与GTP结合的RhoA分子的比例构成了RhoA的“分数激活”，并且是对ROK电位激活的定量度量。在初步工作中，我们构建了一个基于高性能的RhoA激活传感器分子Rhoa.v2。 Rhoa.v2利用mcerulean3和麦克替氏素为定量fret测量提供了出色的特征，尤其是在两光子激发的情况下。两光子激发还提供了在活小鼠完整组织中乃至完全非侵入性的细胞内图像rhoa.v2的能力。 The major Aims of this proposal are to 1) develop a novel transgenic mouse model that expresses RhoA.v2 specifically in smooth muscle cells, 2) develop methods, utilizing two- photon imaging, that unlock the full quantitative power inherent to the design of the RhoA.v2, such that the fractional activation of RhoA can be quantified in arteries in vivo, and 3) pursue a preliminary investigation into the role of Rhoa通过控制动脉中平滑肌细胞收缩的收缩交感神经系统（SNS）活性。 SNS多动症只能存在于活体动物中，是高血压，代谢综合征，心力衰竭和其他疾病的关键因素。我们将检验以下假设：RhoA是体内某些动脉中SNS的关键效应子。这项工作将由一个调查员团队完成，具有光学探针开发/FRET成像（Rizzo博士）和血管生物学和体内成像（Wier博士）方面的专业知识。总而言之，将创建一种新型的RhoA生物传感器小鼠，并利用两光子成像的方法通过亚细胞分辨率开发用于体内RhoA激活的方法。该提案开发的模型和方法将对高血压，糖尿病，许多血管生物学（包括中风）以及一般平滑肌受累的领域（例如胃肠道和膀胱功能）具有广泛的影响。