Pro-Inflammatory Signaling by Endothelial Focal Complexes in Lung

Abstract

Ligation of integrins or subjecting them to mechanical stress induces signaling in intracellular protein complexes known as focal complexes (FC). Such integrin-mediated responses were likely participants in our previously reported finding that the α_vβ₃ integrin increased lung capillary permeability. We identified underlying signaling in bovine pulmonary artery endothelial cell (BPAEC) monolayers exposed to soluble α_vβ₃ ligands. α_vβ₃ ligation induced tyrosine phosphorylation (TyrP) of FC-associated cytoskeletal proteins paxillin, cortactin, and ezrin, as well as the SH2 domain-containing proteins Shc and p125^FAK. During ligation, α_vβ₃ aggregation occurred at the apical surface of endothelial cells (EC). In parallel, α_vβ₃ ligation increased endothelial cytosolic Ca²⁺ concentration ([Ca²⁺]_i), as determined by fura 2 ratio imaging. The [Ca²⁺]_i increase was attributable to release of Ca²⁺ from endosomal stores and Ca²⁺ influx across the plasma membrane. Underlying mechanisms were α_vβ₃-mediated tyrosine phosphorylation of phospholipase C-/gg1 (PLC-/gg1) and production of inositol (1,4,5) trisphosphate (InsP₃). Further studies revealed that α_vβ₃ ligation triggered arachidonate release, which was attributable to the simultaneous increase in [Ca²⁺]_i, cPLA2 (cytosolic phospholipase A2) membrane translocation, and MAPK (mitogen-activated tyrosine kinase) TyrP. Taken together, these findings established the α_vβ₃ integrin in lung EC displayed inflammatory potential in vitro. To understand the relevance of these mechanisms to inflammatory signaling in lung EC in situ, we used a new approach. Using high tidal volume ventilation (HV) in isolated, blood-perfused rat lungs, we subjected EC to mechanical stress. After 2 h, we perfused lungs with collagenase to obtain mixed cells, which we immune sorted (4/dgC) to isolate fresh lung EC (FLEC). Subjection of FLEC lysates to electrophoresis and immunoblotting indicated that HV as compared to low tidal volume ventilation (LV) markedly enhanced TyrP. The tyrosine kinase blocker genistein inhibited this response. Immunofluorescent labeling of FLEC indicated HV induced FC in which aggregates of α_vβ₃ co-localized with FAK. Immunoprecipitation revealed HV-mediated TyrP of the FC protein paxillin, along with paxillin-associated P-selectin expression. Both responses were genistein inhibitable. However, 2-h HV did not increase lung water. These results indicate that in lung EC, the α_vβ₃ integrin forms FC in vitro and in vivo in association with EC TyrP. These responses are associated with [Ca²⁺]_i increase, arachidonate release, and P-selectin expression, effects capable of promoting lung leukocyte sequestration, which further enhances inflammation.