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

描述（由申请人提供）：主要死亡原因，如心脏病、中风和糖尿病，均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了解这些疾病的发病机制和开发新的治疗方法至关重要。许多重要类别的血管调节剂通过与G蛋白偶联受体（GPCR）结合而起作用，所述G蛋白偶联受体（GPCR）启动会聚于小G蛋白RhoA的信号级联。RhoA-GTP激活Rho相关激酶（ROK），其通过抑制肌球蛋白轻链磷酸酶（MLCP）来调节平滑肌的收缩，并且还参与动脉的病理生理学反应;血管重塑、平滑肌细胞增殖和炎性细胞的募集。因此，RhoA可以被认为是一个整合的控制点，将不同的GPCR信号转导转化为许多动脉功能。与GTP结合的RhoA分子的分数构成RhoA的“分数激活”，并且是ROK的潜在激活的定量测量。在初步工作中，我们已经构建了一个高性能的FRET为基础的RhoA激活传感器分子，RhoA.v2。RhoA.v2利用mCerulean 3和mCitrine为定量FRET测量提供出色的特性，特别是双光子激发。双光子激发还提供了在活小鼠的完整组织的细胞内成像RhoA.v2的能力，甚至完全非侵入性地通过皮肤。该提议的主要目的是1）开发在平滑肌细胞中特异性表达RhoA. v2的新型转基因小鼠模型，2）开发利用双光子成像的方法，其解锁RhoA. v2设计所固有的全部定量能力，使得可以在体内动脉中定量RhoA的部分激活，和3）通过以下方法对RhoA在控制动脉平滑肌细胞收缩中的作用进行初步研究： 交感神经系统（SNS）活动。SNS过度活跃，仅存在于活体动物中，是高血压、代谢综合征、心力衰竭和其他疾病的关键因素。我们将测试的假设，RhoA是一个关键的效应SNS在某些动脉在体内。这项工作将由一组在光学探针开发/FRET成像（Rizzo博士）和血管生物学和体内成像（Wier博士）方面具有互补专业知识的研究人员完成。总之，一种新的RhoA生物传感器小鼠将被创建和方法，利用双光子成像，将开发用于定量体内RhoA激活，亚细胞分辨率。该提案开发的模型和方法将对高血压、糖尿病、血管生物学的许多领域（包括中风）以及一般平滑肌参与的领域（如胃肠道和膀胱功能）产生广泛影响。

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.