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（由适用提供）：高血压涉及升高的血管抗性，并且仅存在于活的动物中，其中血管张力的生理调节剂（即神经元活性，血液流动和内分泌因子）完好无损。因此，高血压研究将大大受益于对动脉平滑肌细胞收缩的分子调节剂的体内探索。我们的总体目标是在有意识的动物中，在实验性盐依赖性高血压下首次阐明增加血管抗性的机制。使用有意识的动物（而不是麻醉的动物）是了解交感神经活动（SNA）的推定作用的关键，该作用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无侵入性的，在有意识的光学生物传感器小鼠小动脉中的分子信号传导的荧光成像来实现的。鉴于肌球蛋白调节光链的磷酸化是平滑肌合同的关键确定剂，具体目的1和2是开发出表达新型的，遗传编码的活性生物传感器的光学生物传感器小鼠，用于对平滑肌肌球蛋白磷酸化的平滑肌肉肌球蛋白平滑肌调节剂的关键分子调节剂； A）肌球蛋白轻链激酶（MLCK），B）肌球蛋白轻链磷酸酶，MLCP）和C）MLCP的关键上游调节剂，小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鞘氨醇1-磷酸受体（S1P1）。 SNA对MLCK，MLCP和RHOA的调节将通过在有意识的小鼠中使用完整的自主神经节封锁（Hexamethonium）来确定。小鼠将植入远程伪像，以允许在成像（以及所有其他时间）中连续测量伪造血压。在特定的目标4中，将在有意识的单个小鼠的早期人工伪像（即在血管紧张素的14天内进行一项“纵向'研究），MLCK，MLCP和RHOA的激活水平的时间过程将进行测量 II/盐高血压。小鼠长期注入血管紧张素II并喂养高盐（NACL）饮食。据信，在这种盐依赖性高血压模型中，血管耐药性的增加涉及从对盐敏感的CNS心血管控制区域发出的SNA（``交感神经），强制使用有意识的小鼠。这些具体目标将在两个主要研究人员的指导下在具有必要的专业知识的指导下进行，以生成拟议的传感器（Rizzo）并进行生理实验（wier）。总而言之，我们期望在生物动物中首次发育盐敏感性高血压期间的肌球蛋白辐射调节过程的动态成像。这些研究将揭示有关高血压血管耐药性增加的分子基础的新见解，因为它实际上发生在活动物中。