In PAECs, at the highest dose in particular, the resistance rapidly decreased to ∼60% of baseline, following which there was no further decrease in resistance (Fig. 7A). In PMVECs, at 0.5 and 1.0 U/ml neuraminidase, there was a slow, progressive decrease in resistance until it reached a plateau at 25% baseline value (Fig. 7B). We next treated PAECs and PMVECs with neuraminidase from Clostridium perfringens. Unlike PAECs, at the 2 highest doses of 0.5 and 1.0 U/ml, the resistance progressively decreased to ∼25% of baseline, indicating complete disruption of the endothelial barrier. Similar to what we saw with the PAECs, in neuraminidase-treated PMVECs, staining for α(2,3)-linked sialic acids was still positive, revealing that PMVECs also express a population of neuraminidase-resistant α(2,3)-linked sialic acids (Fig. 6B). Because we observed positive binding of the lectin from Arachis hypogaea following neuraminidase treatment, and because the α(2,3) linkage is the predominant one on PMVECs, it strongly suggests that indeed some α(2,3)-linked sialic acids were cleaved.
C: PAECs (PA) and PMVECs (MV) were treated with 1 U/ml of neuraminidase from Clostridium perfringens. Quantitation of changes in endothelial barrier integrity of PAECs and PMVECs following neuraminidase treatment. PAECs and PMVECs were treated with three different concentrations (0.25 U/ml, 0.5 U/ml, and 1.0 U/ml) of neuraminidase from Vibrio cholerae, and changes in resistance were monitored over 25 h. A: PAECs were treated with neuraminidase from Vibrio cholerae, and changes in resistance were monitored. Thus we conclude that PAECs express at least a subpopulation of neuraminidase-resistant α(2,3)-linked sialic acids. 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure as measured by band intensity on a silver stained gel or other method for determining purity. A: PAECs were treated for 2 h with neuraminidase from Clostridium perfringens (1 U/ml) or Vibrio cholerae (1 U/ml) followed by FITC-tagged SNA to identify the presence of α(2,6)-linked sialic acids. B: PMVECs treated for 2 h with neuraminidase from Clostridium perfringens (1 U/ml) or Vibrio cholerae (1 U/ml) and then treated with FITC-tagged MAA exhibited positive fluorescence, indicating that PMVECs express hydrolytically insensitive α(2,3)-linked sialic acids. D: after 5-h treatment with neuraminidase from Clostridium perfringens or Vibrio cholerae, both PAECs and PMVECs exhibited disruption of the monolayer as evidenced by gap formation.
E: PAECs treated for 2 h with neuraminidase from Clostridium perfringens (1 U/ml) or Vibrio cholerae (1 U/ml) were then treated with FITC-tagged MAA to identify the presence of α(2,3)-linked sialic acids. In these experiments we used a concentration of 1 U/ml of neuraminidase from Clostridium perfringens (Fig. 7C). The PAEC resistance rapidly decreased to ∼75% of baseline after addition of neuraminidase. A: PMVECs were treated for 2 h with neuraminidase from Clostridium perfringens (1 U/ml) or Vibrio cholerae (1 U/ml) followed by FITC-tagged SNA. Lungs treated with 0.5 U/ml neuraminidase from Vibrio cholerae became swollen and edematous (Fig. 8A). The results shown in Fig. 8B indicate that, compared with baseline values, a 30-min neuraminidase treatment caused a severe disruption of the barrier as evidenced by an approximately eightfold increase in permeability (from 0.006 to 0.043 ml × min−1 × cmH20−1 × 100 g−1 of predicted lung weight, respectively). In PMVECs, staining for α(2,6)-linked sialic acids was barely detectable in control cells and was completely absent in neuraminidase-treated cells (Fig. 6A). These results support our earlier observations that PMVECs express little surficial α(2,6)-linked sialic acids. However, at this time we do not know whether one linkage, i.e., α(2,3) or α(2,6), is more important than the other in determining endothelial barrier integrity nor whether further substituted (e.g., acetylated) sialic acids play a role in cell-cell and/or cell matrix adhesion.
