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，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。许多重要的血管调节剂通过与g蛋白偶联受体（gpcr）结合而起作用，gpcr启动信号级联反应，聚集在小GTPase RhoA上。RhoA-GTP激活rho相关激酶（ROK），通过抑制肌球蛋白轻链磷酸酶（MLCP）调节平滑肌收缩，并参与动脉的病理生理反应；血管重构、平滑肌细胞增殖和炎症细胞募集。因此，RhoA可以被视为一个综合控制点，将多种GPCR信号转译为多种动脉功能。RhoA分子与GTP结合的比例构成RhoA的“分数活化”，是衡量ROK潜在活化的定量指标。在前期工作中，我们构建了一个高性能的基于fret的RhoA激活传感器分子，RhoA.v2。RhoA。v2利用mCerulean3和mCitrine为定量FRET测量提供杰出的特性，特别是双光子激发。双光子激发也提供了成像RhoA的能力。V2在活体小鼠的完整组织细胞内，甚至完全非侵入性地通过皮肤。本提案的主要目的是1)开发一种表达RhoA的新型转基因小鼠模型。2)开发方法，利用双光子成像，解锁RhoA设计固有的全部定量能力。(3)对RhoA调控动脉平滑肌细胞收缩的作用进行初步探讨

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.