Creation of Optical Biosensor Mice for Longitudinal Studies of Vascular Function

用于血管功能纵向研究的光学生物传感器小鼠的创建

• 批准号: 9242698
• 负责人: MEGAN A RIZZO
• 金额: $ 38.59万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9242698
• 关键词: Adrenergic Receptor; Angiotensin II; Angiotensin II Receptor; Animals; Arteries; Autonomic ganglion; Biosensor; Blood Pressure; Blood Vessels; Blood flow; Calibration; Calmodulin; Cardiovascular system; Cells; Characteristics; Chronic; Color; Conscious; Coupled; DNA cassette; Data; Development; Diet; Disease; Ear; Endocrine; Endothelin; Endothelin A Receptor; Enzymes; Event; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Goals; Hexamethonium; Hypertension; Image; Implant; Individual; Injection of therapeutic agent; Knowledge; Lead; Ligands; Longitudinal Studies; Measurement; Measures; Methods; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Muscle Cells; Muscle Contraction; Myosin ATPase; Myosin Light Chain Kinase; Myosin Regulatory Light Chains; Nerve; Neurons; Nonmuscle Myosin Type IIA; Operative Surgical Procedures; Optics; Pathway interactions; Pharmacology; Phosphorylation; Physiological; Pressure Transducers; Principal Investigator; Procedures; Process; Reagent; Recovery; Regulation; Reporting; Research; Rho-associated kinase; Role; Rosa; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Sodium Chloride; Sphingosine-1-Phosphate Receptor; Tamoxifen; Techniques; Telemetry; Testing; Time; Tissues; Transgenic Mice; Transgenic Organisms; Vascular Smooth Muscle; Vascular remodeling; Vascular resistance; arteriole; base; constriction; experimental study; fluorescence imaging; genetic regulatory protein; human disease; in vivo; insight; intravital imaging; molecular imaging; myosin phosphatase; novel; novel strategies; public health relevance; quantitative imaging; salt sensitive; salt sensitive hypertension; sensor; sphingosine 1-phosphate

 DESCRIPTION (provided by applicant): Hypertension involves elevated vascular resistance and only exists in a living animal, where the physiologic regulators of vascular tone (i.e. neuronal activity, blood flow, and endocrine factors) are intact. Therefore, hypertension research will greatly benefit from the in vivo exploration of the molecular regulators of arterial smooth muscle cell contraction. Our overall goal is to elucidate mechanisms of increased vascular resistance, for the first time, in conscious animals, during experimental salt-dependent hypertension. The use of conscious animals (as opposed to anesthetized) is key to understanding the putative role of sympathetic nerve activity (SNA), increasingly recognized as a key mechanism of salt-dependent hypertension. This overall goal is to be achieved through the use of non-invasive, fluorescence imaging of molecular signaling in arterioles of conscious optical biosensor mice. Given that phosphorylation of myosin regulatory light chains is the critical determinant of smooth muscle contraction, Specific Aims 1 and 2 are to develop optical biosensor mice that express novel, genetically-encoded activity biosensors for the key molecular regulators of smooth muscle myosin phosphorylation; a) myosin light chain kinase, (MLCK), b) myosin light chain phosphatase, MLCP), and c) a key upstream regulator of MLCP, the small GTPase, RhoA. In Aim 3, we will use these optical biosensor mice to determine, in vivo, 1) the regulation of MLCK, MLCP and RhoA by certain vascular G-protein coupled receptors (GPCR) putatively involved in hypertension, including adrenoceptors (α1-AR), Angiotensin II receptors (AT1-R), endothelin-1 receptors, (ETA), and sphingosine-1-phosphate receptors (S1P1). The regulation of MLCK, MLCP and RhoA by SNA will be determined through the use of complete autonomic ganglionic blockade (hexamethonium) in conscious mice. Mice will be implanted with telemetric arterial blood pressure transducers to allow continuous measurement of arterial blood pressure during imaging (and all other times). In Specific Aim 4, the time course of the activation levels of MLCK, MLCP and RhoA will be measured in ear arterioles of conscious individual mice (i.e. a `longitudinal' study) during 14 days of Angiotensin II/salt hypertension. Mice are infused chronically with Angiotensin II and fed a high-salt (NaCl) diet. Increased vascular resistance in this model of salt-dependent hypertension is believed to involve heightened SNA (`sympathoexcitation) emanating from salt-sensitive CNS cardiovascular control regions, mandating use of conscious mice. These Specific Aims will be performed in this multi-PI project under the direction of two Principal Investigators with the necessary expertise to generate the proposed sensors (Rizzo) and perform the physiologic experiments (Wier). In summary, we expect to achieve dynamic imaging of myosin phosphorylation regulatory processes during the development of salt-sensitive hypertension in a living animal for the first time. These studies will reveal new insights on the molecular basis of increased vascular resistance in hypertension, as it actually occurs in living animals.

 描述（由申请人提供）：高血压涉及血管阻力升高，仅存在于活体动物中，其中血管张力的生理调节因子（即神经元活动、血流和内分泌因子）完好无损。因此，在体探索动脉平滑肌细胞收缩的分子调节因子将极大地有利于高血压的研究。我们的总体目标是阐明在实验性盐依赖性高血压期间，首次在清醒动物中增加血管阻力的机制。使用清醒动物（而不是麻醉动物）是理解交感神经活动（SNA）的假定作用的关键，SNA越来越多地被认为是盐依赖性高血压的关键机制。这一总体目标是通过使用非侵入性的荧光成像的分子信号在有意识的光学生物传感器小鼠的小动脉中实现的。鉴于肌球蛋白调节轻链的磷酸化是平滑肌收缩的关键决定因素，具体目标1和2是开发光学生物传感器小鼠，其表达用于平滑肌肌球蛋白磷酸化的关键分子调节剂的新型遗传编码活性生物传感器; a）肌球蛋白轻链激酶（MLCK），B）肌球蛋白轻链磷酸酶，MLCP），和c）MLCP的关键上游调节物，小GT3，RhoA。目的3：利用光学生物传感器检测1）高血压相关血管G蛋白偶联受体（GPCR）puroneceptors，包括肾上腺素受体（α1-AR）、血管紧张素Ⅱ受体（AT 1-R）、内皮素受体（ETA）和1-磷酸鞘氨醇受体（S1 P1）对MLCK、MLCP和RhoA的调节。将通过在清醒小鼠中使用完全自主神经节阻滞（六烃季铵）来确定SNA对MLCK、MLCP和RhoA的调节。小鼠将植入遥测动脉血压传感器，以允许在成像期间（和所有其他时间）连续测量动脉血压。在特定目标4中，在14天的血管紧张素A治疗期间，将在清醒个体小鼠的耳小动脉中测量MLCK、MLCP和RhoA的活化水平的时间过程（即“纵向”研究）。 II/盐性高血压。小鼠长期输注血管紧张素II并喂食高盐（NaCl）饮食。盐依赖性高血压模型中血管阻力的增加被认为涉及源自盐敏感性CNS心血管控制区的SNA（“交感神经兴奋"）的增强，这要求使用清醒的小鼠。这些特定目标将在本多PI项目中在两名主要研究者的指导下执行，该主要研究者具有生成申报传感器（Rizzo）和执行生理实验（Wier）所需的专业知识。总之，我们期望首次在活体动物中实现盐敏感性高血压发展过程中肌球蛋白磷酸化调节过程的动态成像。这些研究将揭示高血压血管阻力增加的分子基础的新见解，因为它实际上发生在活体动物中。