HIF-1 is an ab-heterodimer: the HIF-1b subunit is constitutively nuclear whereas HIF-1a is inducible by hypoxia [7]. Regulation of HIF-1a protein level involves its posttranslational hydroxylation at proline residues 402 and 564 (human sequence) by prolylhydrox- ylases (PHDs), predominantly PHD2 [8]. PHDs are Fe(II)- and 2- oxoglutarate-dependent dioxygenases that have an absolute re- quirement for molecular oxygen [9]. In oxygenated cells, proline hydroxylations target HIF-1a to the von Hippel-Lindau protein complex for poly-ubiquitylation followed by proteasome-dependent degradation [10]. Under hypoxia, HIF PHDs are inactivated, HIF- 1a escapes proteolytic degradation, migrates to the cell nucleus, and binds to HIF-1b and co-factors to initiate the transcription of target genes. These target genes include glucose transporters, most glycolytic enzymes, the lactate transporter monocarboxylate transporter 4 (MCT4) and key pro-angiogenic effectors such as vascular endothelial growth factor (VEGF) [11,12]. Because HIF-1 promotes both glycolytic energy production and angiogenesis, it is not surprising that increased levels of HIF-1a are associated with poor cancer prognosis [13,14]. Several pieces of evidence suggest that the glycolytic switch triggers angiogenesis [15]. The most direct coupling is probably exerted by lactate, the end-product of glycolysis. Lactate has been known for many years to promote angiogenesis in wounds. It promotes collagen deposition [16,17] and enhances VEGF production and activity in fibroblasts [18] and macrophages [19]. The underlying mechanism has been proposed to involve lactate oxidation to pyruvate, together with a decrease in NAD+ and a subsequent decrease in protein poly-ADP-ribosylation [17– 20]. In glioma tumor cells, lactate was further shown to trigger HIF-1 activation in a hypoxia-independent manner through inhibition of HIF-1a proline hydroxylations [21,22], resulting in increased VEGF production by tumor cells. A direct impact of lactate on HIF-1a expression in endothelial cells has however not been documented. We recently reported that lactate could stimulate nuclear factor-kappa B (NF-kB) activation and interleukin-8 (IL-8) production in ECs [23], proving that these cells could act as a lactate signaling platform continuously fueled by the elevated lactate concentration (1–40 mM) present in the tumor microenvironment [24]. In this study, we report that lactate stimulates HIF-1 activity in ECs, which directly accounts for angiogenesis independently of any additional pro-angiogenic stimulus. We further identify the lactate transporter monocarbox- ylate transporter 1 (MCT1) as the main regulator of HIF-1 activation by lactate in ECs, and provide unprecedented evidence that HIF-1 inhibition and the associated blockade of angiogenesis may be obtained through MCT1 inhibition. Results ECs take up exogenous lactate Confluent ECs were exposed to 10 mM sodium L-lactate, a concentration that corresponds to the level of lactate most commonly detected in human tumors [25]. 13C-lactate (methyl- labeled) was used in nuclear magnetic resonance (NMR) experiments. 13C NMR spectra of cell lysate collected 6-h after treatment showed a peak at 19 ppm corresponding to intracellular 13C-lactate (Figure 1A). In the same conditions, 1H NMR spectra showed doublets arising from intracellular 13C-lactate at 1.56 and 1.30 ppm, thus confirming that ECs import exogenous lactate (Figure 1B). The uptake rate was quantified in an enzymatic assay specific for L-Lactate. Kinetic analyses revealed a time-dependent increase in intracellular lactate after exposure to 10 mM extracellular lactate (Figure 1C). Targeting MCT1 Blocks Tumor Angiogenesis Exogenous lactate activates HIF-1 in normoxic ECs Lactate is known to fuel oxidative phosphorylation in tumor cells and favors cell proliferation even in the absence of an exogenous source of serum or growth factors [26]. In similar serum-free conditions, however, lactate cannot rescue glucose- deprived ECs as documented by an accelerated increase in cell death (Figure S1A). In the presence of glucose, it did not modulate the oxygen consumption rate (Figure S1B). In the following experiments, we therefore examined the signaling effects of lactate added to the growth medium of ECs and we focused on HIF-1, based on previous reports in tumor cells [21,22]. Using a dual luciferase reporter assay in normoxic ECs, we found increased HIF-1 activity after a 24-h treatment with 10 mM lactate (Figure 2A). Consistent with HIF-1 activation, we evidenced the nuclear translocation of HIF-1a (Figure S2). VEGF and basic fibroblast growth factor (bFGF) are major pro-angiogenic agents [27]. VEGF-A is encoded by a HIF-1 target gene [28] while bFGF expression is indirectly induced by HIF-1 [29]. To further investigate HIF-1 activation by lactate, we probed changes in VEGF-A and bFGF release in the supernatant of confluent human umbilical vein endothelial cells (HUVECs) using ELISA. VEGF-A levels were unchanged (Figure 2B) but a 3-fold increase in bFGF secretion was detected after a 24-h incubation with 10 mM lactate (Figure 2C). The same treatment significantly increased the protein expression of VEGF receptor-2 (VEGFR2/flk-1) (Figure 2D), the major transducer of VEGF-mediated angiogenesis in ECs [27]. Increased membrane VEGFR2 expression was confirmed using specific immunostaining in HUVECs (Figure S3A). PCR arrays further revealed that lactate upregulated the transcription of 31 genes in HUVECs by at least ,2-fold, of which 10 are known to be direct HIF-1 target genes (Table S1). HIF-1 activity primarily depends on the stable expression of its a subunit [7]. In normoxic HUVECs, lactate (10 mM) induced a significant increase in HIF-1a protein expression 24-h after treatment (Figure 3A). Similar induction was observed in bovine aortic ECs (BAECs) (Figure 3B). Induction of HIF-1a by lactate was concentration-dependent with a plateau corresponding to a 3- fold increase in HIF-1a protein expression reached after a 24-h exposure to lactate concentrations $10 mM (Figure 3C). It was also time-dependent with a 5-fold induction peak at 3-h followed by a slow decrease (Figure 3D). The canonical regulation of HIF-1 activity involves the posttranslational hydroxylation of HIF-1a on 2 proline residues, a reaction catalyzed by HIF PHDs that promotes HIF-1a degradation [8]. Our observations that lactate did not significantly alter HIF-1a transcription (Figure 4A) and still induced HIF-1a protein expression in the presence of the transcription inhibitor Actinomycin D (Figure 4B) prompted us to check whether lactate could interfere with the PHD reaction. Considering that PHD activity requires 2-oxoglutarate as a substrate and may therefore be influenced by other carboxylates [21–23], we designed competition experiments between lactate and 2-oxoglutarate in normoxic ECs. Addition of 2-oxoglutarate to ECs concentration- dependently reduced the abundance of HIF-1a in ECs exposed to lactate (Figure 4C; a representative blot is shown in Figure S4A). We also used an ODD-Luc construct to detect PHD activity [30] and showed that 10 mM lactate triggered ODD-driven luciferase activity as efficiently as 150 mM CoCl2 (Figure 4D). We next aimed to determine whether lactate or pyruvate (resulting from intracellular lactate oxidation) exerted these effects on PHD activity. Pyruvate cannot be detected using NMR. Therefore, we developed an enzymatic assay to show that exposure of ECs to exogenous lactate led to a time-dependent increase in intracellular pyruvate (Figure 4E). Lactate oxidation to PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33418