Function of the 130kDa MLCK in vasculature physiology and pathophysiology

130kDa MLCK 在脉管系统生理学和病理生理学中的功能

• 批准号: 7372166
• 负责人: BRIAN Paul HERRING
• 金额: $ 34.24万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7372166
• 关键词: Ablation; Alleles; Angiotensin II; Animals; Arteries; Asthma; Atherosclerosis; Biological Assay; Blood Pressure; Blood Vessels; Cardiovascular system; Cell Culture Techniques; Cell Proliferation; Cell physiology; Cells; Data; Development; Disease; Elements; Endothelial Cells; Fluorescein; Fluoresceins; Functional disorder; Gene Proteins; Genes; Goals; Hypertension; In Vitro; Injury; Introns; Ischemia; Knock-out; Knockout Mice; Laboratories; Life; Limb structure; Measurement; Modeling; Mus; Myosin Heavy Chains; Myosin Light Chain Kinase; Pathology; Permeability; Physiological; Physiology; Play; Process; Property; Protein Isoforms; Proteins; Regulatory Element; Research; Role; Site; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Stimulus; System; Testing; Vascular Diseases; Vascular Endothelial Cell; Vascular System; angiogenesis; cell motility; design; embryonic stem cell; femoral artery; homologous recombination; in vivo; insight; matrigel; migration; promoter; protein expression; research study; response; restenosis; telokin

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the physiological and pathological roles of the different MLCK isoforms, encoded by the mylk1 gene, in the vasculature. The mylk1 gene encodes a 220kDa myosin light chain kinase (MLCK), a 130kDa MLCK, as well as the non-catalytic gene product telokin. Experiments described in this proposal are designed to test the hypothesis that the 130kDa MLCK plays a specific non- redundant role in regulating smooth muscle contractility and endothelial cell function in the vascular system. We will generate mice harboring a smooth muscle or endothelial cell-specific knockout of the 130kDa MLCK isoform, while maintaining expression of the other mylk1 gene products. To do this we will use a loxP/cre system to delete key regulatory elements within the 130kDa MLCK promoter. Analysis of the properties of the smooth muscle and endothelial cells in these animals under normal and pathological conditions will allow us determine the specific functions of the 130kDa MLCK in vivo. Two specific aims are proposed to achieve these goals. In aim 1 we will generate mice in which LoxP sites flank the core of the 130kDa MLCK promoter or a conserved element within intron 1. The LoxP mice will be crossed with smooth muscle MHC- cre/eGFP mice to facilitate deletion of the regulatory elements, and subsequent ablation of 130kDda MLCK expression, specifically in differentiated smooth muscle cells. Telemetric measurements of blood pressure will be made in live animals to assess changes in vascular tone in 130kDa MLCK knockout mice. Smooth muscle tissues will be isolated from these mice and their contractile properties determined in detail, ex vivo. MLCK has been implicated in regulating cell migration and proliferation of many cells, including smooth muscle cells. We will, therefore, also examine the effects of the ablation of the 130kDa MLCK on pathological models that induce smooth muscle cell migration and proliferation. A wire induced injury of the mouse femoral artery and collateral artery remodeling following hind limb ischemia will be used. In our second aim we will cross the 130kDa MLCK LoxP mice with Tie-2 cre mice to result in the knockout of the 130kDa MLCK specifically in endothelial cells. The subsequent effects on endothelial barrier function in vivo will be assayed by fluorescein permeability assays. Changes in angiogenesis will be assayed in a matrigel plug assay and in a mouse hind limb ischemia model. Together these data will provide a comprehensive analysis of the roles played by the 130kDa MLCK in the vasculature under physiological and pathophysiological conditions.

描述（由申请人提供）：本研究的长期目标是确定由mylk1基因编码的不同MLCK亚型在脉管系统中的生理和病理作用。mylk1基因编码一个220kDa的肌球蛋白轻链激酶（MLCK），一个130kDa的MLCK，以及非催化基因产物端粒蛋白。本文所述的实验旨在验证130kDa MLCK在调节血管系统中平滑肌收缩力和内皮细胞功能方面发挥特定的非冗余作用的假设。我们将产生具有平滑肌或内皮细胞特异性敲除130kDa MLCK亚型的小鼠，同时保持其他mylk1基因产物的表达。为此，我们将使用loxP/cre系统删除130kDa MLCK启动子中的关键调控元件。分析这些动物在正常和病理条件下的平滑肌和内皮细胞的特性，将使我们能够确定130kDa MLCK在体内的具体功能。为实现这些目标，提出了两个具体目标。在目标1中，我们将产生LoxP位点位于130kDa MLCK启动子核心侧面或内含子1内保守元件的小鼠。LoxP小鼠将与平滑肌MHC- cre/eGFP小鼠杂交，以促进删除调控元件，并随后消融130kDda MLCK表达，特别是在分化的平滑肌细胞中。将在活体动物中进行血压遥测测量，以评估130kDa MLCK基因敲除小鼠血管张力的变化。将从这些小鼠身上分离出平滑肌组织，并在体外详细测定其收缩特性。MLCK参与调节包括平滑肌细胞在内的许多细胞的迁移和增殖。因此，我们还将研究130kDa MLCK消融对诱导平滑肌细胞迁移和增殖的病理模型的影响。采用后肢缺血后小鼠股动脉钢丝损伤及侧支动脉重构的方法。在我们的第二个目标中，我们将130kDa MLCK LoxP小鼠与Tie-2 cre小鼠杂交，从而在内皮细胞中特异性地敲除130kDa MLCK。随后对体内内皮屏障功能的影响将通过荧光素渗透性测定来测定。血管生成的变化将在基质栓试验和小鼠后肢缺血模型中进行分析。这些数据将全面分析130kDa MLCK在生理和病理生理条件下在脉管系统中所起的作用。