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

描述(申请人提供)：主要死因，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。许多重要的血管调节剂通过与G蛋白偶联受体(GPCRs)结合来发挥作用，GPCRs启动信号级联，汇聚到小GTP酶RhoA上。RhoA-GTP通过抑制肌球蛋白轻链磷酸酶(MLCP)激活Rho相关激酶(ROK)，调节血管收缩，并参与动脉的病理生理反应、血管重塑、平滑肌细胞增殖和炎性细胞募集。因此，RhoA可以被认为是一个整合的控制点，将不同的GPCR信号转化为多种动脉功能。与GTP结合的RhoA分子的分数构成了RhoA的“部分激活”，是对韩国潜在激活的定量衡量。在前期工作中，我们构建了一个基于FRET的高性能RhoA激活传感器分子RhoA.v2。RhoA.v2利用mCerulean3和mCitine为定量FRET测量提供卓越的特性，特别是在双光子激发下。双光子激发还提供了在活着的小鼠完整组织的细胞内成像RhoA.v2的能力，甚至是完全非侵入性的，通过皮肤。这一建议的主要目的是：1)建立一种新的转基因小鼠模型，在平滑肌细胞中特异性地表达RhoA.v2；2)利用双光子成像，开发方法，解锁RhoA.v2设计所固有的全部定量力量，使得RhoA的部分激活可以在活体动脉中定量；以及3)初步探讨RhoA在控制动脉平滑肌细胞收缩中的作用。 交感神经系统(SNS)活动。仅存在于活体动物中的SNS过度活动是高血压、代谢综合征、心力衰竭和其他疾病的关键因素。我们将在体内验证RhoA在某些动脉中是SNS的关键效应者的假设。这项工作将由一组研究人员完成，他们在光学探头开发/FRET成像(Rizzo博士)、血管生物学和活体成像(Wier博士)方面拥有丰富的专业知识。总之，将创建一种新型的RhoA生物传感器小鼠，并将开发利用双光子成像的方法，以亚细胞分辨率在体内定量RhoA的激活。这一建议开发的模型和方法将广泛影响高血压、糖尿病、血管生物学的许多领域(包括中风)，以及一般平滑肌受累的领域，如胃肠道和膀胱功能。

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.