INFLAMMATORY MECHANISMS IN LUNG VASCULAR SEGMENTS

肺血管段的炎症机制

基本信息

批准号：
6927826
负责人：
KAUSHIK PARTHASARATHI
金额：
$ 40.35万
依托单位：
ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-22 至 2007-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6927826
关键词：
arterioles calcium flux capillary cell component structure /function confocal scanning microscopy fluorescent dye /probe free radical oxygen immunofluorescence technique inflammation ionophores laboratory rat leukocyte activation /transformation lung mitochondria morphometry tumor necrosis factor alpha vascular endothelium

项目摘要

DESCRIPTION (provided by applicant): Aims: Our overall objective is to understand the regulation of pro-inflammatory responses that develop in a spatially distributed, segment specific manner in the intact lung capillaries. Here, we will determine the role of mitochondrial mechanisms in regulation of leukocyte margination in arteriolar, septal and venular capillaries. The specific aims are to quantify for the first time capillary segment specific, regulation of endothelial (EC) mitochondrial (Ca2+mit) and cytosolic Ca2+cyt) (Specific Aim 1), generation of EC mitochondrial reactive oxygen species (ROS) (Specific Aim 2), and mitochondria-mediated leukocyte margination (Specific Aim 3). The new hypotheses to be tested are that mitochondrial distribution and the induction of mitochondrial mechanisms are vascular segment specific, and that differences in the extent to which mitochondrial mechanisms are invoked determine differences in pro-inflammatory responses in the various vascular segment of the lung. Procedures: 1) Morphometric measurements. Intravital imaging of mitochondrial and endosomal stores (ER) will be conducted in capillaries of the isolated, blood-perfused rat lung, using both conventional and confocal microscopy. 2) Ca2+ quantification, Ca2+mit, Ca2+cyt, and ER Ca2+ changes will be determined using flurophores that target the appropriate compartment. 3) ROS quantification. EC ROS production will be determined using fluorometric imaging of the ROS indicator dichloro fluorescin using our reported protocols. In one set of experiments we will test the hypotheses in a mouse lacking components of NADPH-oxidase to determine the role on non-mitochondiral ROS production. 4) Immunofluorescence imaging. Expression of P-selectin in capillaries will be determined using indirect in situ immunofluorescence imaging. 5) Leukocyte margination. Leukocyte margination will be determined using confocal and conventional microscopy using leukocytes labeled with, rhodamine 6G. 6) Ca2+ increase. EC Ca2+ will be increased by a) infusion of agonists and b) in situ photo-excited intracellular uncaging. Responses will be determined in terms of mitochondrial mechanisms that increase ROS production and leukocyte margination. Significance: This proposal addresses a new understanding of pro-inflammatory responses in different capillary segments. These segment-specific responses, which determine the preferential pathway for leukocyte margination during inflammation, are not adequately understood. It is important to understand the role of mitochondria, as mitochondrial mechanisms and mitochondrial ROS may critically regulate leukocyte margination and hence, lung injury. Mitochondrial ROS may also be involved in signaling gene transcription and consequently, lung remodeling. If the preliminary data hold, then this research will prove for the first time that mitochondrial mechanisms regulate vascular segment specific pro-inflammatory responses.

描述（由申请人提供）：目的：我们的总体目标是了解以完整的肺毛细血管以空间分布的特定方式发展的促炎反应的调节。在这里，我们将确定线粒体机制在小动脉，隔膜和静脉毛细血管中的白细胞边缘调节中的作用。具体的目的是定量首次特定毛细管段特异性，内皮（EC）线粒体（CA2+MIT）和胞质CA2+CA2+CAC2）（特定目的1），EC线粒体活性氧（ROS）（ROS）（特定的目标2），以及线粒体介导的Leukcocy Tecipy（特定目标）（特定目标）。要测试的新假设是线粒体分布和线粒体机制的诱导是血管段的特异性，并且在调用线粒体机制的程度上差异决定了肺部各个血管段中炎性反应的差异。程序：1）形态测量值。线粒体和内体储存（ER）的插入成像将使用常规和共聚焦显微镜在分离的，血液中的大鼠肺的毛细血管中进行。 2）CA2+定量，CA2+ MIT，Ca2+ Cyt和ER Ca2+更改将使用针对适当隔室的flurophores确定。 3）ROS定量。 EC ROS的产生将使用我们报告的方案使用ROS指示剂二氯荧光素的荧光成像来确定。在一组实验中，我们将测试缺乏NADPH-氧化酶成分的小鼠中的假设，以确定在非局部性病性ROS产生中的作用。 4）免疫荧光成像。 P-选择素在毛细血管中的表达将使用间接原位免疫荧光成像确定。 5）白细胞边缘。白细胞边缘将使用标记为Rhodamine 6g的白细胞使用共焦和常规显微镜确定。 6）CA2+增加。 EC CA2+将通过a）输注激动剂和b）原位光兴奋的细胞内未约会来增加。响应将根据线粒体机制来确定，从而增加ROS产生和白细胞边缘。意义：该提案介绍了对不同毛细管段中促炎反应的新理解。这些特异性反应确定了炎症期间白细胞边缘的优先途径，尚未充分了解。重要的是要了解线粒体的作用，因为线粒体机制和线粒体ROS可能会严重调节白细胞边缘，因此肺损伤。线粒体ROS也可能参与信号基因转录，因此，肺重塑。如果有初步数据，那么这项研究将首次证明线粒体机制调节血管段特定的促炎反应。