Shear Stress Activation of AMPK Cascade: Implications in Vascular Biology

AMPK 级联的剪切应力激活：对血管生物学的影响

基本信息

批准号：
7487698
负责人：
Lemar Irvin Smith
金额：
$ 2.96万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7487698
关键词：
5&apos -AMP-activated protein kinase AMP-activated protein kinase kinase Adherent Culture Adipose tissue Affect Animal Model Anti-Inflammatory Agents Anti-inflammatory Apolipoprotein E Apoptotic Area Arterial Fatty Streak Arteries Atherosclerosis Automobile Driving Biological Biological Assay Biological Availability Biological Models Biology Biomechanics Blood Vessels Bos taurus Brain Breeding Ca(2+)-Calmodulin Dependent Protein Kinase Calcium/calmodulin-dependent protein kinase Cardiovascular system Cattle Causations Cell Line Cell physiology Chemicals Copy Number Polymorphism Crossbreeding Diabetes Mellitus Diagnosis Dominant-Negative Mutation Endothelial Cells Engineering Epigenetic Process Exercise Frequencies Functional disorder Gene Expression Gene Targeting Genes Goals Heart Homeostasis Hormones Human Hypertension Hypoxia Investigation Knock-out Knockout Mice Liver Maintenance Mechanics Metabolic Metabolism Modification Molecular Mus Mutant Strains Mice Nature Nitric Oxide Nitric Oxide Synthase Nutrient Patients Pattern Phosphotransferases Physiological Play Post-Translational Protein Processing Prevention Production Proteins Research Resistance Role STK11 gene Signal Transduction Skeletal Muscle Small Interfering RNA Specimen Stimulus Surface Testing Tissue-Specific Gene Expression Tissues Transcriptional Activation Transcriptional Regulation Variant Vascular Endothelial Cell Vasodilation Western Blotting Wild Type Mouse Work atheroprotective base cell type chromatin immunoprecipitation hemodynamics in vitro Model in vivo inhibitor/antagonist insight loss of function monolayer research study response shear stress size transcription factor

项目摘要

DESCRIPTION (provided by applicant): The broad long term objective/goal of the proposed work is to gain insight into the mechanical and molecular mechanisms by which vascular endothelial cells (ECs) sense hemodynamic stimuli and transduce the resulting signals to post-translational modifications and transcriptional regulation. Three specific aims are proposed to test the hypothesis that AMP-activated protein kinase (AMPK) in ECs is activated by atheroprotective flow, which in turn upregulates genes beneficial to vascular tone. Specific Aim 1: to delineate the biomechanical basis of atheroprotective flow in activating the AMPK kinase (AMPK)-AMPK cascade in ECs; Specific Aim 2: to elucidate the molecular basis underlying the transcriptional activation of atheroprotective genes by AMPK in response to atheroprotective flow; and Specific Aim 3: to investigate the ro|e of flow activated AMPK in vascular tone ex vivo and atheroprotection in vivo. We will use flow channels to subject a monolayer of cultured ECs to flow with variations in hemodynamic factors (magnitude, temporal/spatial gradients, frequency and amplitude) and assess the activation of AMPKK, namely camodulin kinase kinase and LKB1, and AMPK (by kinase activity assay and Western blot) as biological readouts. We will then determine the molecular basis underlying the differential gene expression (e.g. eNOS, ASS, ACC, CNP) in ECs responding to different flows. We will utilize chromatin immunoprecipitation (ChIP) assays to study the epigenetic modification of the AMPK target genes. Parallel inhibition experiments utilizing siRNA, dominant negative mutant of AMPK, and pharmalogical inhibitors will be conducted to provide supporting evidence. We will investigate gene expression pattern, NO bioavailability, and EC- dependent vascular tone as functional consequences. The use of selected breeding of gene specific (i.e., ampk-/-, apoE-/- mice) mutant mice will allow us to assess distribution and extent of atherosclerotic lesions are enhanced because of the loss-of-function of AMPK. This project will help to define the parameters and thresholds distinguishing atheroprotective from atheroprone flow patterns. Study of the molecular mechanisms by which the defined flow patterns affect vasodilation and endothelial function will contribute to defining the mechanical and chemical factors involved in causation/prevention/diagnosis/treatment of endothelial dysfunction (e.g. atherosclerosis, hypertension, diabetes).

描述（由申请人提供）：拟议工作的广泛长期目标/目标是深入了解血管内皮细胞（ECS）感知血液动力学刺激并转导的机械和分子机制，并将结果信号转换为后翻译后的修饰和转录调节。提出了三个特定的目的来检验以下假设：ECS中AMP激活的蛋白激酶（AMPK）被动脉保护流激活，这反过来又上调了对血管张力的有益基因。具体目的1：描述激活ECS中AMPK激酶（AMPK）-AMPK CASCADE的动脉保护流的生物力学基础；特定目的2：阐明AMPK响应动脉保护流的AMPK对动脉保护基因的转录激活的基础分子基础；和特定目的3：研究血管张力的流动AMPK在体内的流动AMPK和动脉保护性。我们将使用流动通道对培养的EC的单层进行单层，以随着血液动力学因子的变化（时间/空间/空间梯度，频率和振幅）的变化，并评估AMPKK的激活，即Camodulin激酶激酶激酶和LKB1和LKB1，LKB1，LKB1和AMPK（通过激酶活性测定和蛋白质读数）。然后，我们将确定差异基因表达（例如eNOS，ASS，ACC，CNP）的分子基础，对不同的流动响应。我们将利用染色质免疫沉淀（CHIP）测定来研究AMPK靶基因的表观遗传修饰。利用siRNA，AMPK的显性阴性突变体和药物抑制剂的平行抑制实验将进行提供支持证据。我们将研究基因表达模式，无生物利用度和依赖性血管张力作为功能后果。由于AMPK的功能丧失，因此使用了基因特异性（即AMPK - / - ，APOE-/ - 小鼠）突变小鼠的选定育种将使我们能够评估动脉粥样硬化病变的分布和程度。该项目将有助于定义参数和阈值，从而区分动脉op剂与动脉op骨流动模式。对定义的流动模式影响血管舒张和内皮功能的分子机制的研究将有助于定义参与因果关系/预防/诊断/治疗内皮功能障碍的机械和化学因素（例如，动脉粥样硬化，高血压，高血压，糖尿病）。