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

描述（由申请人提供）：导致死亡的主要原因，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) 结合来发挥作用，GPCR 启动信号级联，汇聚到小 GTP 酶 RhoA 上。 RhoA-GTP 激活 Rho 相关激酶 (ROK)，该激酶通过抑制肌球蛋白轻链磷酸酶 (MLCP) 调节平滑肌收缩，并且还参与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，可将不同的 GPCR 信号转译为多种动脉功能。与 GTP 结合的 RhoA 分子的分数构成了 RhoA 的“激活分数”，并且是 ROK 潜在激活的定量测量。在前期工作中，我们构建了一种基于 FRET 的高性能 RhoA 激活传感器分子 RhoA.v2。 RhoA.v2 利用 mCerulean3 和 mCitrine 为定量 FRET 测量（尤其是双光子激发）提供出色的特性。双光子激发还提供了对活体小鼠完整组织细胞内的 RhoA.v2 进行成像的能力，甚至可以完全非侵入性地通过皮肤进行成像。该提案的主要目的是 1) 开发一种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，解锁 RhoA.v2 设计固有的全部定量能力，以便可以量化体内动脉中 RhoA 的激活分数，以及 3) 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、心力衰竭等疾病的关键因素。我们将检验 RhoA 是体内某些动脉中 SNS 的关键效应器的假设。这项工作将由在光学探针开发/FRET 成像（Rizzo 博士）以及血管生物学和体内成像（Wier 博士）方面拥有互补专业知识的研究人员团队完成。总之，将创建一种新型 RhoA 生物传感器小鼠，并开发利用双光子成像的方法，以亚细胞分辨率定量体内 RhoA 激活。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（例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.