Figure 3

Analysis of VEGF-D expression. (a–c) Representative double immunofluorescent staining for VEGFR-3 and LYVE-1 (a), and for CD68 and VEGF-D (b) in the diaphragm of a methylglyoxal mouse is shown. Arrows and arrowheads of the same color indicate the same cells. Scale bars, 50 μm. Quantification of CD68- and VEGF-D-stained cells (number of labeled cells per high power field (HPF)) in control and methylglyoxal mouse models is shown in c. VEGF-D was mainly expressed by CD68-positive macrophages in the methylglyoxal mouse model (c). n=6 in both control and methylglyoxal models. (d and e) VEGF-D mRNA (d) and protein (e) expression analyzed using RT–PCR and ELISA, respectively, were not detected in the human mesothelial cell line Met5A (d, lane 1) (e), in mesothelial cells derived from the peritoneal dialysis effluent of the patients on peritoneal dialysis (d, lane 2) (e), or in mesothelial cells from the omentum (d, lane 3) (e). In contrast, VEGF-D mRNA was detected in peritoneal tissues (d, lane 4) and VEGF-D protein was detected in peritoneal dialysis effluent (e) from patients with ultrafiltration failure (Table 1). (d) GAPDH was used as a loading control. PD, peritoneal dialysis, ND, not detected.