Characterization of CD24 as a novel lymphatic valve marker in human and mouse. (A and B) Whole mounts prepared from the upper 200 µm of human dermis, showing the expression of CD24 at valve-like regions in PDPN+ LVs. The picture shown in B represents a Tilescan of multiple stitched images. Scale bars: left, 50 µm; right, 100 µm. Representative images from >10 independent experiments (donors). (C and D) Flow cytometry–based analysis of CD24 expression in LYVE-1low and LYVE-1high LECs (CD45−CD31+PDPN+) in the stromal vascular fraction of human dermis. (C) Representative histogram plot showing the gating of LYVE-1low and high LECs. (D) Quantification of the percentage (%) of CD24-expressing LECs in LYVE-1low and LYVE-1high LECs from three independent experiments (n = 3 donors). Each dot represents one independent experiment. Statistics were computed with paired Student’s t test. *P < 0.05. (E) CD24 expression in mouse LN LECs. Representative flow cytometry plots of three experiments. (F) LN LECs were subjected for 48 h to laminar flow (Lam, 4 dyn/cm2) or static conditions. RT-qPCR was performed on the extracted mRNA. The fold change is shown, and values above 1 represent genes upregulated under shear stress. Each dot represents one independent experiment performed with LN LECs from different isolations (n = 3). (G and H) Whole mounts of Prox1-eGFP mouse mesentery showing expression of (G) CD24 in lymphatic valves visualized with Prox1 (high in valves) and (H) in combination with the cell adhesion molecule CD31. Representative images from five independent experiments (G and H). Scale bars: (G) 150 µm, (H) 50 µm. (I and J) Quantification of mesenteric valves in Cd24−/− or WT pups. In each experiment, mesenteries were collected from 4- to 5-day-old littermate pups (WT Cd24+/+ and Cd24−/−) obtained from Cd24+/− × Cd24+/− crosses. Valves were quantified using whole-mount histology. (I) Representative images of WT and Cd24−/− mesenteries showing VEGFR3 staining. Some valves are indicated by a red arrow. Note that for the identification of valves, the combination of several markers was used (see Materials and methods). Scale bar: 2 mm. (J) Quantification of the valve numbers in mesenteric vessels in WT (+/+) and Cd24−/− mesenteries. The absolute number of valves per mm LV is shown. In each experiment, all WT (+/+) and Cd24−/− mesenteries from littermates of a Cd24+/− × Cd24+/− crossing were analyzed. In total, pups from six litters containing at least one pup from each genotype were analyzed. Each dot represents the value from one pup. Statistics: linear mixed-effects model with litter as a random effect to account for within- and between-litter variation. ***P < 0.001.
Figure 7.

Characterization of CD24 as a novel lymphatic valve marker in human and mouse. (A and B) Whole mounts prepared from the upper 200 µm of human dermis, showing the expression of CD24 at valve-like regions in PDPN+ LVs. The picture shown in B represents a Tilescan of multiple stitched images. Scale bars: left, 50 µm; right, 100 µm. Representative images from >10 independent experiments (donors). (C and D) Flow cytometry–based analysis of CD24 expression in LYVE-1low and LYVE-1high LECs (CD45−CD31+PDPN+) in the stromal vascular fraction of human dermis. (C) Representative histogram plot showing the gating of LYVE-1low and high LECs. (D) Quantification of the percentage (%) of CD24-expressing LECs in LYVE-1low and LYVE-1high LECs from three independent experiments (n = 3 donors). Each dot represents one independent experiment. Statistics were computed with paired Student’s t test. *P < 0.05. (E) CD24 expression in mouse LN LECs. Representative flow cytometry plots of three experiments. (F) LN LECs were subjected for 48 h to laminar flow (Lam, 4 dyn/cm2) or static conditions. RT-qPCR was performed on the extracted mRNA. The fold change is shown, and values above 1 represent genes upregulated under shear stress. Each dot represents one independent experiment performed with LN LECs from different isolations (n = 3). (G and H) Whole mounts of Prox1-eGFP mouse mesentery showing expression of (G) CD24 in lymphatic valves visualized with Prox1 (high in valves) and (H) in combination with the cell adhesion molecule CD31. Representative images from five independent experiments (G and H). Scale bars: (G) 150 µm, (H) 50 µm. (I and J) Quantification of mesenteric valves in Cd24−/− or WT pups. In each experiment, mesenteries were collected from 4- to 5-day-old littermate pups (WT Cd24+/+ and Cd24−/−) obtained from Cd24+/− × Cd24+/− crosses. Valves were quantified using whole-mount histology. (I) Representative images of WT and Cd24−/− mesenteries showing VEGFR3 staining. Some valves are indicated by a red arrow. Note that for the identification of valves, the combination of several markers was used (see Materials and methods). Scale bar: 2 mm. (J) Quantification of the valve numbers in mesenteric vessels in WT (+/+) and Cd24−/− mesenteries. The absolute number of valves per mm LV is shown. In each experiment, all WT (+/+) and Cd24−/− mesenteries from littermates of a Cd24+/− × Cd24+/− crossing were analyzed. In total, pups from six litters containing at least one pup from each genotype were analyzed. Each dot represents the value from one pup. Statistics: linear mixed-effects model with litter as a random effect to account for within- and between-litter variation. ***P < 0.001.

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