Aims: Our objective is to understand lung endothelial signaling mechanisms that underlie lung injury induced by the ventilation stress. High volume mechanical ventilation is used in the management of respiratory failure and often causes inflammatory consequences in the lung. Although lung endothelial cells are likely to underlie these responses, relevant cell signaling mechanisms are not known. In preliminary data we show that ventilation stress induces tyrosine phophorylation-dependent proinflammatory signaling in lung endothelial cells. We will better define this response in the context of focal adhesion formation (Aim 1), expression of leukocyte adhesion receptors on the endothelial surfaces (Aim 2), and leukocyte-endothelial interactions (Aim 3). Procedures: Experiments will be conducted using our newly developed methods involving freshly isolated lung endothelial cells (FLEC). Isolated, blood-perfused rat lungs will be ventilation stressed by mechanically ventilating them in different groups exposed to variations in tidal volume, breathing frequency, stress duration and end-expiratory pressure. FLEC will be recovered from stressed and control lungs. Immunofluorescence studies on whole cells and immunoblotting and immunoprecipitation studies on cell lysates will be conducted to determine tyrosine phosphorylation of focal adhesion proteins, cPLA2 activation, Jun kinase activation and expression of leukocyte adhesion receptors. RT-PCR studies will be conducted to determine c-jun mRNA expression. Significance: These experiments will provide the first understanding of lung endothelial signaling induced by ventilation stress. Endothelial signaling is understood largely from in vitro studies in cultured cells. Our project will provide novel understanding of signaling mechanism relevant to endothelial cells in vivo. Specifically, we will clarify the fundamental question of whether focal adhesion formation in mechanical stress drives lung endothelial signaling towards proinflammatory pathways. These mechanisms are highly relevant to the general understanding of the lung inflammatory response. No previous understanding of these mechanisms exists for lung endothelium in vivo. The proposed studies will be novel.
