Targeting MCT1 Blocks Tumor Angiogenesis Figure 5. Targeting MCT1 inhibits lactate-induced HIF-1 activation in normoxic endothelial cells. (A) Relative mRNA expression was determined in confluent HUVECs using quantitative RT-PCR. n = 3. Error bars reflect mean 6 SEM. (B) Immunostaining showing the membrane expression of MCT1 in HUVECs (upper panel) and negative control (lower panel, omission of primary antibody). Bar = 40 mm. (C) HUVECs transfected with the indicated siRNA were incubated during 24-h with 10 mM lactate or not. HIF-1a protein expression was detected by western blot. *p,0.05, ***p,0.005 compared to control; ###p,0.005 compared lactate alone; n = 6–14. (D and E) HIF-1a and VEGFR2 protein expression was detected by western blot. (D) HUVECs were incubated during 24-h with 10 mM lactate and/or 5 mM a-cyano-4-hydroxycinnamate (CHC). *p,0.05, **p,0.01, ***p,0.005 compared to control; ###p,0.005 compared to lactate alone; n = 3–12. (E) BAECs were incubated during 24-h with 10 mM lactate, 5 mM Nv-nitro-L-arginine methyl ester (L-NAME), 5 mM a-cyano-4-hydroxycinnamate (CHC), and/or 5 mM N-acetyl-L-cysteine (NAC). *p,0.01, ***p,0.005 compared to control; ###p,0.005 compared to lactate alone; n = 3–14. (C–E) Error bars reflect mean 6 SEM. doi:10.1371/journal.pone.0033418.g005 and the MCT1 inhibitor CHC (Figure 7B). Using undiluted Matrigel, we demonstrated that MCT1 silencing with a specific shRNA not only inhibited lactate-induced tubulogenesis but also basal tubulogenesis (Figure S8), suggesting autocrine lactate signaling in cultured ECs. We performed in vivo experiments to validate lactate as a pro- angiogenic agent and MCT1 as a new anti-angiogenic target. First, Matrigel plugs were implanted subcutaneously in each flank of mice, the left plug containing 10 mM lactate and the right plug an equal volume of saline. Seven days later, CD31 immunolabel- ing of collected plugs revealed a ,10-fold increase in the endothelial colonization of lactate-containing plugs compared to controls (Figure 8A). Most vascular structures adopted a circular shape and were perfused (Figure 8A, inset), thus confirming lactate PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33418