Aims: The overall objective is to understand the in situ cell biology of lung endothelial cells. Here, The PI will determine the role of the endothelial cytosolic calcium concentration (EC[Ca2+]i) in proinflammatory responses in lung microvessels induced by sustained pressure stress.
The specific aims are to define in lung microvessels: 1) the regulation of EC[Ca2+]i); 2) the regulation of leukocyte entrapment; 3) the generation of diffusible second messengers; and 4) the distribution of proinflammatory mRNA. The new hypotheses to be tested are that in lung microvessels, sustained pressure-stress causes: 1) increased expression of leukocyte adhesion receptors; 2) increased leukocyte entrapment; and 3) increase production of reactive oxygen species (ROS) and NO. Procedures: The isolated, blood-perfused rat lung will be used. Pressure-stress will be established by increasing the left atrial pressure for set periods. Arteriolar, septal and venular capillaries will be imaged by digital fluorescence microscopy. In these vessels, in situ quantifications will be obtained of EC[Ca2+]i by the fura 2-ratio method, EC exocytosis as determined by the fluorescence of FM1-43 at fusion-pores, ROS by the H202-sensitive dye DCFH and [NO] (NO concentration) by a potentiometric method. In addition, expression of leukocyte adherence receptors on the EC surface of lung capillaries will be determined by in situ immunoimaging. To determine the extent and within-capillary distribution of gene transcription responses, lung capillaries will be subjected to in situ PCR for detection of mRNA of selected leukocyte adhesion receptors and chemokines. Significance: Sustained high pressure is well known to be pathogenic in lung microvessels. However, relevant cellular mechanisms are not well understood. It is important to know the role of EC[Ca2+]i in this biology since high pressure increases EC[Ca2+]i and induces Ca2+ dependent expression of the leukocyte adhesion receptor, P-selection in lung venular capillaries. Increased EC[Ca2+]i may be common to many mechanisms that promote lung microvascular injury. Sustained EC[Ca2+]i increases, or EC[Ca2+]i oscillations in pathological conditions, may induce secondary effects such as generation of other second messengers and induction of gene transcription and consequently, lung vascular remodeling. The proposed studies are therefore novel and important, and will reveal a fundamental, new understanding of pressure-induced responses in the lung capillary.
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