Expression of ESM-1 in LECs is induced by VEGF-A and VEGF-C via VEGFR-2 and VEGFR-3. (A) Quantitative RT-PCR (qPCR) analysis revealed that compared with untreated controls, LECs expressed levels of ESM-1 mRNA that were more than 10-fold higher after 24 hours of stimulation with VEGF-A (●), and more than 4-fold higher after 24 hours of stimulation with VEGF-C (■); these findings confirmed those of the microarray expression results (○, □) (B) Immunoblot analysis confirmed that LECs stimulated with VEGF-A for 24 or 48 hours expressed much higher levels of ESM-1 in the cell lysates and in cell supernatants compared with untreated controls (0 hours). β-actin levels were used as gel loading controls. (C) Treatment of LECs with VEGF-A compared with PBS strongly induced the expression of ESM-1 in the presence of control antibody (IgG). ESM-1 induction was completely inhibited by a VEGFR-2–blocking antibody (αR2). Incubation with a VEGFR-3–blocking antibody (αR3) partially reduced the induction of ESM-1 by VEGF-A, and a combination of both blocking antibodies (αR2 + αR3) inhibited the VEGF-A–mediated ESM-1 induction. (D) Compared with PBS, VEGF-C also induced the expression of ESM-1. Incubation with either an αR2 or αR3 only partially blocked the ESM-1 induction by VEGF-C; ESM-1 induction by VEGF-C was completely prevented by a combination of both receptors. ***P < .001; **P < .005; *P < .01. Error bars represent standard deviation. The LYVE-1⁺ lymphatic vessels in the skin of wild-type mice did not express ESM-1 (E-G), whereas the LYVE-1⁺ lymphatic vessels of VEGF-A transgenic mice (H-J) did express ESM-1 (panel J; arrows). Scale bars equal 100 μm.