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

基本信息

项目摘要

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mylk 1基因编码的不同MLCK亚型在血管系统中的生理和病理作用。mylk 1基因编码一个220 kDa的肌球蛋白轻链激酶(MLCK),一个130 kDa的MLCK,以及非催化基因产物telokin。本提案中描述的实验旨在检验130 kDa MLCK在调节血管系统中的平滑肌收缩性和内皮细胞功能中发挥特定的非冗余作用的假设。我们将产生具有130 kDa MLCK亚型的平滑肌或内皮细胞特异性敲除的小鼠,同时维持其他mylk 1基因产物的表达。为此,我们将使用loxP/cre系统删除130 kDa MLCK启动子内的关键调控元件。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130 kDa MLCK在体内的特定功能。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130 kDa MLCK启动子核心或内含子1内保守元件侧翼的小鼠。LoxP小鼠将与平滑肌MHC-cre/eGFP小鼠杂交,以促进调控元件的缺失,以及随后的130 kDda MLCK表达的消除,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130 kDa MLCK敲除小鼠中血管张力的变化。将从这些小鼠中分离平滑肌组织,并离体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细胞迁移和增殖。因此,我们还将研究消融130 kDa MLCK对诱导平滑肌细胞迁移和增殖的病理模型的影响。将使用金属丝诱导的小鼠股动脉损伤和后肢缺血后的侧支动脉重塑。在我们的第二个目标中,我们将使130 kDa MLCK LoxP小鼠与Tie-2 cre小鼠杂交,以导致内皮细胞中特异性敲除130 kDa MLCK。将通过荧光素渗透性测定法测定对体内内皮屏障功能的后续影响。将在基质胶塞测定和小鼠后肢缺血模型中测定血管生成的变化。总之,这些数据将提供一个全面的分析所发挥的作用,130 kDa MLCK在脉管系统的生理和病理生理条件下。

项目成果

期刊论文数量(1)
专著数量(0)
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会议论文数量(0)
专利数量(0)

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BRIAN Paul HERRING其他文献

BRIAN Paul HERRING的其他文献

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{{ truncateString('BRIAN Paul HERRING', 18)}}的其他基金

Regualtion of visceral smooth muscle-specific gene expression during development.
发育过程中内脏平滑肌特异性基因表达的调节。
  • 批准号:
    7895243
  • 财政年份:
    2009
  • 资助金额:
    $ 37.55万
  • 项目类别:
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
  • 财政年份:
    2009
  • 资助金额:
    $ 37.55万
  • 项目类别:
Synthetic smooth muscle cell-selective promoters
合成平滑肌细胞选择性启动子
  • 批准号:
    6701180
  • 财政年份:
    2004
  • 资助金额:
    $ 37.55万
  • 项目类别:
Synthetic smooth muscle cell-selective promoters
合成平滑肌细胞选择性启动子
  • 批准号:
    6848778
  • 财政年份:
    2004
  • 资助金额:
    $ 37.55万
  • 项目类别:
Synthetic smooth muscle cell-selective promoters
合成平滑肌细胞选择性启动子
  • 批准号:
    7017007
  • 财政年份:
    2004
  • 资助金额:
    $ 37.55万
  • 项目类别:
Visceral smooth muscle-specific gene expression
内脏平滑肌特异性基因表达
  • 批准号:
    6446620
  • 财政年份:
    2001
  • 资助金额:
    $ 37.55万
  • 项目类别:
Visceral smooth muscle-specific gene expression
内脏平滑肌特异性基因表达
  • 批准号:
    6524795
  • 财政年份:
    2001
  • 资助金额:
    $ 37.55万
  • 项目类别:
Visceral smooth muscle-specific gene expression
内脏平滑肌特异性基因表达
  • 批准号:
    6791250
  • 财政年份:
    2001
  • 资助金额:
    $ 37.55万
  • 项目类别:
Visceral smooth muscle-specific gene expression
内脏平滑肌特异性基因表达
  • 批准号:
    6933179
  • 财政年份:
    2001
  • 资助金额:
    $ 37.55万
  • 项目类别:
Regualtion of visceral smooth muscle-specific gene expression during development.
发育过程中内脏平滑肌特异性基因表达的调节。
  • 批准号:
    7628017
  • 财政年份:
    2001
  • 资助金额:
    $ 37.55万
  • 项目类别:

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