OPTICAL IMAGING OF LUNG CAPILLARIES IN VIVO

描述（由申请人提供）： 目的：我们的总体目标是了解在完整的肺毛细血管中以空间分布、节段特异性方式发展的促炎反应的调节。在这里，我们将确定在不同深度的肺胸膜表面在体内毛细血管的这种调节。具体来说，我们将测试的假设，线粒体机制调节肺泡隔毛细血管白细胞边缘。具体目标是首次在胸膜不同深度的毛细血管中定量内皮（EC）线粒体（Ca 2 +mit）和胞质Ca 2+（Ca 2 +cyt）（具体目标1）的调节、EC线粒体活性氧（ROS）（具体目标2）的产生和ESTA介导的白细胞边缘化（具体目标3）。 步骤：（1）形态测量。采用双光子显微镜对大鼠肺毛细血管线粒体和内体钙库进行活体成像。(2)Ca 2+定量。将使用靶向适当隔室的荧光团测定Ca 2 +mit、Ca 2 +cyto和ER Ca 2+变化。(3)ROS定量。EC ROS生产将使用ROS指示剂二氯荧光素的荧光成像，使用我们报告的协议来确定。(4)免疫荧光成像。将使用间接原位免疫荧光成像来确定毛细血管中P-选择素的表达。(5)白细胞边缘化。将使用罗丹明6 G标记的白细胞的双光子显微镜测定白细胞边集和迁移。(6)Ca 2+升高。（a）激动剂的输注和（B）原位光激发的细胞内游离可增加EC Ca 2+。将根据增加ROS产生和白细胞边缘化的线粒体机制来确定应答。 意义：该提案提出了对胸膜不同深度毛细血管中肺部炎症生物学的新认识。该计划是在单个内皮细胞原位水平上实现对肺毛细血管反应的全面理解。理解线粒体的作用是重要的，因为线粒体机制和线粒体ROS可以关键地调节白细胞边缘化，从而调节肺损伤。线粒体ROS也可能参与信号基因转录，从而参与肺重塑。如果初步数据成立，那么这项研究将首次证明线粒体机制调节特定的促炎反应。

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 this regulation in capillaries at different depths from the pleural surface of the lung in vivo. Specifically, we will test the hypothesis that mitochondrial mechanisms regulate leukocyte margination in alveolar septal capillaries. The specific aims are to quantify for the first time in capillaries at different depths from the pleura, regulation of endothelial (EC) mitochondrial (Ca2+mit) and cytosolic Ca 2+ (Ca2+cyt) (Specific Aim 1), generation of EC mitochondrial reactive oxygen species (ROS) (Specific Aim 2), and mitochondria-mediated leukocyte margination (Specific Aim 3). Procedures: (1) Morphometric measurements. Intravital imaging of mitochondrial and endosomal Ca 2+ stores (ER) will be conducted in capillaries of the rat lung, using two-photon microscopy. (2) Ca2+quantification. Ca2+mit, Ca2+cyto and ER Ca 2+ changes will be determined using fluorophores 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. (4) Immunofluorescence imaging. Expression of P-selectin in capillaries will be determined using indirect in situ immunofluorescence imaging. (5) Leukocyte margination. Leukocyte margination and migration will be determined using two photon microscopy of leukocytes labeled with, rhodamine 6G. (6) Ca 2+ increase. EC Ca 2+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 the biology of lung inflammation in capillaries at different depths from the pleura. The plan is to achieve global understanding of lung capillary responses at the level of the single endothelial cell in situ. 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 specific pro-inflammatory responses.