Signaling Induced by Inflammatory Mediators in the Rodent Pulmonary Microvasculature

Acute lung inflammation (ALI), stemming from a disproportionate and detrimental immune response, may arise from or complicate other disease states, leading to the often-fatal acute respiratory distress syndrome (ARDS). Because of the many culpable factors and differing points of induction, pinning down the signaling mechanisms involved in the morbidity of this disorder as well as defining an effective treatment has proved problematic. However, the most detrimental characteristic of this condition is seen regardless of the development of the response: increased microvascular permeability. Because of the architecture and the size of the pulmonary microvascular network, the lungs have a resident, sequestered population of leukocytes that are able to rapidly respond to injury or infection, but may also contribute to the pathology of ALI/ARDS by increasing endothelial barrier dysfunction. Many inflammatory mediators dictate the course and gravity of the response by inducing endothelial cytoskeletal reorganization, such as induction of actin stress fibers, cell rounding and contraction, and dissociation of interendothelial junctions. Thrombin is a well-studied mediator that has been shown to be barrier-disruptive rapidly increases microvascular permeability. Sphingosin-1-phosphate (S1P) is a more novel, less understood mediator that has been shown to mediate basal vascular permeability as well as to enhance barrier integrity in inflammation. Inflammatory signaling may also expand throughout the lungs through intercellular communication via gap junctions composed of connexins, such as Connexin 43 (Cx43). Herein, we explored how intercellular communication through Cx43-containing gap junctions mediates thrombin-induced signaling as well as the interplay between thrombin- and S1P- induced signaling on the pulmonary microvascular barrier. We isolated and perfused lungs from rats and mice, a physiologically-relevant model to study lung inflammation. We found that focal micropuncture instillations of thrombin were able to induce responses related to hyperpermeability (including, changes in intracellular Ca2+, increased F-actin polymerization, and increased reactive oxygen species generation) both in microvessels directly treated with thrombin and those far outside the instilled region (up to 1000 μm away), and the expansion of signaling into the untreated microvessels was due to intercellular communication mediated by Cx43. For the F-actin polymerization response, we determined that the specific second messenger being communicated and propagating the thrombin-induced increase was inositol trisphosphate (IP3). We also found that, though thrombin induced increases in mean Ca2+in cultured cells, it instead induced increases in the amplitude of cytosolic Ca2+ oscillations in pulmonary microvessels. While we observed that untreated primary pulmonary microvascular endothelial cells and pulmonary microvessels from mice lacking endothelial Cx43 displayed higher levels of the S1P receptor, S1P2, thrombin induced an increase in S1P2 expression that was dependent on the presence of Cx43. While S1P itself was able to partially rescue barrier integrity following thrombin treatment, we show for the first time that S1P2 signaling substantially contributes to thrombin-induced endothelial hyperpermeability, and that inhibiting S1P2 significantly reduced thrombin-induced permeability increases.