Sialic acids play an important role in the protection of PDPN from proteolytic degradation. (A) Flow cytometric analyses of cell surface PDPN on WT or C1galt1^−/− ECs with or without sialidase treatment. Cells were incubated with hamster anti-PDPN antibody 8.1.1, followed by PE-conjugated anti-hamster IgG. (B) Glycan profile of WT PDPN-eGFP or core 1 O-glycan–deficient PDPN-GFP with or without sialidase treatment. WT PDPN-eGFP or core 1 O-glycan–deficient PDPN-eGFP was purified by mouse anti-GFP monoclonal antibody agarose beads from WT ECs or C1galt1^−/− ECs. The fluorescence intensity of anti-PDPN antibody or lectin binding is indicated with bars. Lectin specificities are as follows: LFA (sialic acid); MAL-II (Siaα2-3Galβ1-3 ± [Siaα2-6]GalNAc); sambucus nigra agglutinin (Siaα2-6Gal or Siaα2-6GalNAc); PNA (desialylated Galβ1-3GalNAc [core 1]); HPA (terminal GalNAc [Tn]); and RCA120 (desialylated Galβ1-4GlcNAc [LacNAc]). The data represent the mean ± SD from 3 experiments. (C) WT ECs were pretreated with sialidase and then incubated in buffer (as a control), or 2 μg/ml activated MMP-2/MMP-9 with or without 100 μM GM6001. The cells were stained with anti-PDPN antibody for flow cytometry. The bar graph indicates the percentage of MFI of cell surface PDPN levels against that of control. The data represent the mean ± SD from 3 experiments. (D) Sialidase activity was measured using RCA120 reactivity with fetuin treated by culture medium, the lymph from sham, the lymph from CLP mice, or the lymph from CLP mice with 300 μg/ml of sialidase inhibitor at 37°C for 2 hours. The fluorescence intensity of RCA120 binding is indicated with bars. The data represent the mean ± SD from 3 experiments. (E) WT ECs were incubated in culture medium (as a control), lymph from sham, or lymph from CLP mice with or without 100 μM GM6001, 100 μM GM6001 and 2 mM PMSF, or 300 μg/ml sialidase inhibitor. The bar graph indicates the percentage of MFI of cell surface PDPN levels against that of control. The data represent the mean ± SD from 3 experiments. (F, top) Flow cytometric analyses of cell surface PDPN on isolated mesenteric LECs from mice treated by sham with vehicle, CLP with vehicle, CLP with GM6001, or CLP with sialidase inhibitor. PDPN expression was analyzed on CD31⁺/Lyve-1⁺ LEC population. (F, bottom) Quantification of the percentages of PDPN⁺ LEC numbers (left) and percentages of MFIs of PDPN on LECs (right) are shown. MFIs of PDPN on LECs from CLP mice were normalized to the MFI of PDPN on LECs from sham mice. Data represent the mean ± SD from 4 experiments. *P < .05; **P < .01; ***P < .001. ND, not detected.