MMPs in the lymph cleave core 1 O-glycan–deficient PDPN in vitro and in vivo. (A) Stable PDPN-eGFP WT or C1galt1^−/− ECs were incubated in culture medium (as a control), lymph only, lymph with a 1:100 diluted protease inhibitor cocktail, with 5 mM EDTA, or with 100 mM GM6001, and then labeled with anti-PDPN antibody for flow cytometry. The bar graph indicates the percentage of MFI of cell surface PDPN levels against that of control. Data represent the mean ± SD from 3 experiments. (B) Lymph from C57BL/6J mice was analyzed for MMP-2 and MMP-9 by gelatin zymography. Purified mouse pro–MMP-2 and recombinant mouse pro–MMP-9 were activated by APMA and used as positive controls. Samples were resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing 1 mg/mL gelatin, then visualized by staining with Coomassie Brilliant Blue G-250. (C) Confocal images of intestinal cryosections from vehicle-treated WT, vehicle-treated EHC C1galt1^−/−, or GM6001-treated EHC C1galt1^−/− mice stained with antibodies against Lyve-1, Tn, and PDPN. Scale bar, 20 μm. (D, top) Flow cytometric analyses of cell surface PDPN on freshly isolated mesenteric LECs from vehicle-treated WT, vehicle-treated EHC C1galt1^−/−, or GM6001-treated EHC C1galt1^−/−mice. PDPN expression was analyzed on the CD31⁺/Lyve-1⁺ LEC population. (D, 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 vehicle-treated EHC C1galt1^−/− or GM6001-treated EHC C1galt1^−/− mice were normalized to the MFI of PDPN on LECs from vehicle-treated WT mice. Data represent the mean ± SD from 5 experiments. *P < .05; **P < .01; ***P < .001.