Platelets enhance endothelial adhesiveness in high tidal volume ventilation.

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 P-selectin-blocking antibody in the lung perfusion. The expression was also blocked in lungs from P-selectin knockout (P sel(-/-)) mice perfused with autologous blood, but not with heterologous wild-type 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 HV-induced 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.

Sequence of endothelial signaling during lung expansion.

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.

High tidal volume ventilation induces proinflammatory signaling in rat lung endothelium.

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.

Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice.

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in microl h(-1) cm(-2), n = 9 mean+/-s.e.): 4.3+/-0.6, wildtype mice (MCE); 3.5+/-0.6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in microl h(-1) cm(-2)) induced by a 100 mOsm sucrose gradient across MCE cell layers (8.7+/-0.6, n = 8) was significantly greater than that across KMCE (5.7+/-0.7, n = 6, p = 0.007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in microm sec(-1): 74+/-4, n = 19 vs. 44+/-4 microm sec(-1), n = 11, p < 0.001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99+/-1, n = 19 vs. KMCE: 64+/-5, n = 11, p < 0.001). Thus, as in other 'low rate' fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium.

Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells.

The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.