Alveolar overdistension during mechanical ventilation causes leukocyte sequestration, leading to lung injury. However, underlying endothelial cell (EC) mechanisms are undefined. In a new approach, we exposed isolated blood-perfused rat lungs to high tidal volume ventilation (HV) for 2 h, then obtained fresh lung endothelial cells (FLEC) by immunosorting at 4 degrees C. Immunoblotting experiments indicated that as compared with FLEC derived from lungs ventilated at low volume (LV), HV markedly enhanced tyrosine phosphorylation (TyrP). The tyrosine kinase blocker, genistein, inhibited this response. HV also induced focal adhesion (FA) formation in FLEC, as detected by immunofluorescent aggregates of the alpha(v)beta(3) integrin that co-localized with aggregations of focal adhesion kinase (FAK). Immunoprecipitation and blotting experiments revealed that HV increased TyrP of the FA protein, paxillin. In addition, HV induced a paxillin-associated P-selectin expression on FLEC that was also inhibited by genistein. However, HV did not increase lung water. These results indicate that in HV, EC signaling in situ causes FA formation and induces TyrP-dependent P-selectin expression. These signaling mechanisms may promote leukocyte-mediated responses in HV.

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Although platelets induce lung inflammation, leading to acute lung injury (ALI), the extent of platelet-endothelial cell (EC) interactions remains poorly understood. Here, in a ventilation-stress model of lung inflammation, we show that platelet-EC interactions are important. We obtained freshly isolated lung endothelial cells (FLECs) from isolated, blood-perfused rat lungs exposed to ventilation at low tidal volume (LV) or stress-inducing high tidal volume (HV). Immunofluorescence and immunoprecipitation studies revealed HV-induced increases in cell-surface von Willebrand factor (vWf) expression on FLEC. This increased expression was inhibited by platelet removal from the lung perfusion and by including a Pselectin-blocking antibody in the lung perfusion. The expression was also blocked in lungs from P-selectin knockout (P sel 2/2 ) mice perfused with autologous blood, but not with heterologous wildtype blood containing P-selectin-expressing platelets. These findings indicate that in ventilation stress, platelets transfer vWf to the EC surface and that platelet P-selectin plays a critical role in this transfer. Further evidence for such intercellular transfers was the HVinduced FLEC expressions of platelet glycoprotein 1b and of platelet P-selectin. We conclude that in ventilation stress, platelets deposit leukocyte-and platelet-binding proteins on the EC surface, thereby establishing the proinflammatory phenotype of the vascular lining.

Although high tidal volume ventilation exacerbates lung injury, the mechanisms underlying the inflammatory response are not clear. Here, we exposed isolated lungs to high or low tidal volume ventilation, while perfusing lungs with whole blood, or blood depleted of leukocytes and platelets. Then, we determined signaling responses in freshly isolated lung endothelial cells by means of immunoblotting and immunofluorescence approaches. In depleted blood perfusion, high tidal volume induced modest increases in both P-selectin expression on the endothelial surface, and in endothelial protein tyrosine phosphorylation. Both high tidal volume-induced responses were markedly enhanced in the presence of whole blood perfusion. However, a P-selectin-blocking antibody given together with whole blood perfusion inhibited the responses down to levels corresponding to those for depleted blood perfusion. These findings indicate that the full proinflammatory response occurs in two stages. First, lung distension causes modest endothelial activation. Second, subsequent endothelial-inflammatory cell interactions augment P-selectin expression and tyrosine phosphorylation. We conclude that interactions of circulating inflammatory cells with P-selectin critically determine proinflammatory endothelial activation during high tidal volume ventilation.Keywords: leukocytes; lung; mechanical; P-selectin; phosphorylation Although mechanical ventilators provide essential respiratory support in lung injury, a potential difficulty is that the high tidal volumes delivered by mechanical ventilation may independently exacerbate lung injury (1). Since high tidal volume (HV) causes excessive expansion of pulmonary alveoli, and since stretching cultured endothelial cells activates kinases (2), it is proposed that alveolar overexpansion stretches endothelial cells, causing inflammatory responses. However, no direct data in situ support this view.Because endothelial cells are critical for initiating lung inflammation, it is important to understand the endothelial signaling sequence activated by HV ventilation. An important but unanswered question is whether lung expansion is the predominant factor that induces proinflammatory endothelial signaling, or whether secondary inputs are required. The stretch hypothesis is derived largely from findings in cultured cells (2). Hence, the possibility exists that even under HV conditions, lung endothelial cells in situ may not stretch sufficiently to account entirely for the induced inflammatory consequences.To test this hypothesis, we exposed lungs to HV ventilation. Our aim was to determine the extent to which alveolar stretch, (Received in original form April 11, 2005 and in final form July 19, 2005) This study was supported by grant HL54157 to S.B. from the National Institutes of Health.Correspondence and requests for reprints should be addressed to Sunita Bhattacharya, M.D., St. Luke's-Roosevelt Hospital Center, AJA #510, 1000 10th Avenue, New York, NY 10019. E-mail: sb80@columbia.edu as opposed to inflammato...

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