OPTICAL IMAGING OF LUNG CAPILLARIES IN VIVO

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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.