Aims: The overall objective of the proposed studies is to understand the regulation of proinflammatory responses that develop in a spatially distributed, segment specific manner in the intact lung microvasculature. In this proposal, we will determine the role of connexin-dependent intercellular communication in the induction of microvascular responses in lung disease. In the proposed specific aims, we will quantify for the first time vascular segment specific, connexin-dependent spatial spread of endothelial calcium increases (Aim 1), induction of proinflammatory responses (Aim 2), and regulation of intercellular communication in lung disease (Aim 3). We will test the new hypothesis that differences in connexin-regulation invoke differences in lung pro-inflammatory responses, and that connexin-regulation is vascular segment specific. In addition, we will test the new hypothesis that lung inflammation downregulates intercellular communication as part of the recovery process. Procedures: The general approach in this proposal will be to inhibit intercellular communication and then determine calcium communication, reactive oxygen species production, expression of leukocyte adhesion molecules, and leukocyte recruitment. Moreover, the extent of intercellular communication is modified in lung disease will be defined. We will inhibit intercellular communication by blocking gap junctions and connexin expression using pharmacological and molecular mechanisms. We will determine intercellular calcium increases by our novel photolysis method. We will determine connexin regulation of microvascular function in individual microvascular segments. Relevance. Acute lung injury, which has a high mortality rate in patients, is attributable to a rapid development of inflammation across the lung's vast vascular surface. Currently, no specific mechanism explains this extensive inflammatory spread. This proposal addresses a new understanding for the development of spatially extensive inflammation. Connexin-dependent intercellular communication of calcium may induce reactive oxygen species generation, expression of leukocyte adhesion molecules, and thus leukocyte recruitment and lung injury. However, in lung disease, the recovery may involve downregulation of intercellular communication along with other proinflammatory factors. If our preliminary data hold, then our research will prove for the first time that connexins play a critical role in the spatial spread of inflammation. No previous understanding of these mechanisms exists, and hence, the proposed studies are outstandingly novel and important. PROJECT NARRATIVE: Acute lung injury (ALI) currently has high mortality rate. Our proposal addresses mechanisms that lead to the development of spatially extensive inflammation associated with ALI. Our planned studies will provide a greater understanding of the intercellular communication in lung arterioles, capillaries and venules, and thus lead to development of new therapies for treatment of ALI. ? ? ?
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