ESM-1 promotes LEC proliferation and migration induced by VEGF-A and VEGF-C. (A) Addition of ESM-1 together with VEGF-A (20 ng/mL) or VEGF-C (100 ng/mL) significantly increased the stimulatory effects of both growth factors on LEC proliferation with a minimal effective concentration of 0.01 μg/mL, whereas treatment with ESM-1 alone had no effect. (B) Transfection of LECs with ESM-1 siRNAs reduced ESM-1 protein levels (right lane) compared with control siRNA–transfected LEC (left lane). (C) The proliferation-inducing effects of 30 ng/mL VEGF-A and of 200 ng/mL VEGF-C were suppressed in ESM-1 siRNA–transfected LECs but not in control siRNA–transfected LECs. Addition of 1 μg/mL of ESM-1 promoted LEC proliferation (+14% for VEGF-A, P < .001; +14% for VEGF-C, P = .006). Addition of 1 μg/mL ESM-1 protein to ESM-1 siRNA–transfected LECs partially restored the induction of proliferation by VEGF-A and VEGF-C. (D) Monolayer wound healing assay: ESM-1 (1 μg/mL) promoted the promigratory effect of VEGF-A in a monolayer wound assay after 24 hours (+14% increase in cell migration; P = .012), whereas LECs transfected with ESM-1 siRNA showed a significantly reduced migratory response to VEGF-A compared with control (siRNA-transfected) LECs. At the 48-hour time point, both VEGF-A–treated cultures and cultures treated with VEGF-A plus ESM-1 already showed a 100% wound closure. Addition of 1 μg/mL ESM-1 protein to ESM-1 siRNA–transfected LECs significantly restored the effect of VEGF-A on LEC migration after 24 and 48 hours. *P < .05; **P < .005; ***P < .001. Error bars represent standard deviation.