Platelets Enhance Endothelial Adhesiveness in High Tidal Volume Ventilation

Abstract

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.

Soluble P-Selectin and the Risk of Primary Graft Dysfunction After Lung Transplantation

“…and reconstituted with bone marrow from P-selectin knockout mice (thus permitting endothelial P-selectin but not megakaryocyte P-selectin expression) demonstrated better gas exchange and decreased neutrophil accumulation in the intravascular, interstitial, and intraalveolar spaces of the lung compared to wild-type mice with intact bone marrow. Another study 27 showed that platelet P-selectin was critical to the proinflammatory phenotype of endothelial dysfunction induced by high-tidal volume ventilation. We have previously shown 18 that human lung transplantation itself is associated with platelet activation, even in the absence of traditional risk factors such as significant pulmonary hypertension and CPB.…”

Erythrocytes Induce Proinflammatory Endothelial Activation in Hypoxia

“…Our previous findings indicate that in lungs of Hb mut mice, hypoxia increases erythrocyteendothelial H 2 O 2 transfer as well as Ca 21 -dependent endothelial P-selectin expression (20). Here, we exposed IPLs of Hb mut mice to hypoxia while perfusing the lungs with Hb mut or wildtype (WT) erythrocytes.…”

“…FLECs were isolated on ice (21). Briefly, to clear blood, lungs were perfused with 3 ml of 0.1% BSA-containing PBS.…”

“…The nonimmune fluorescence provided a background for the sample. Cells were stained with fluorophorelabeled antibodies (Alexa 633, Alexa 488, and FITC) under permeabilized (0.1% Triton-X), or nonpermeabilized conditions to enable intracellular or surface staining, respectively (21). Flow cytometry (FACScalibur; Beckton Dickinson, San Jose, CA) gating conditions were set against isotype-matched and fluorophore-matched nonimmune IgG.…”

“…Whether these erythrocyte effects are severe enough to cause pulmonary edema also remains unclear. To address these issues, we determined hypoxic responses in freshly recovered lung endothelial cells (FLECs) that reflect endothelial responses in situ (21). Our findings indicate that erythrocytes are critical for hypoxia-induced NF-kB activation in lung, providing the first evidence, to our knowledge, that hypoxia-induced paracrine effects between erythrocytes and lung endothelium activate proinflammatory responses.…”