Context-dependent regulation of endothelial cell metabolism: differential effects of the PPARβ/δ agonist GW0742 and VEGF-A.

Peroxisome proliferator activated receptor β/δ (PPARβ/δ) has pro-angiogenic functions, but whether PPARβ/δ modulates endothelial cell metabolism to support the dynamic phenotype remains to be established. This study characterised the metabolic response of HUVEC to the PPARβ/δ agonist, GW0742, and compared these effects with those induced by VEGF-A. In HUVEC monolayers, flux analysis revealed that VEGF-A promoted glycolysis at the expense of fatty acid oxidation (FAO), whereas GW0742 reduced both glycolysis and FAO. Only VEGF-A stimulated HUVEC migration and proliferation whereas both GW0742 and VEGF-A promoted tubulogenesis. Studies using inhibitors of PPARβ/δ or sirtuin-1 showed that the tubulogenic effect of GW0742, but not VEGF-A, was PPARβ/δ- and sirtuin-1-dependent. HUVEC were reliant on glycolysis and FAO, and inhibition of either pathway disrupted cell growth and proliferation. VEGF-A was a potent inducer of glycolysis in tubulogenic HUVEC, while FAO was maintained. In contrast, GW0742-induced tubulogenesis was associated with enhanced FAO and a modest increase in glycolysis. These novel data reveal a context-dependent regulation of endothelial metabolism by GW0742, where metabolic activity is reduced in monolayers but enhanced during tubulogenesis. These findings expand our understanding of PPARβ/δ in the endothelium and support the targeting of PPARβ/δ in regulating EC behaviour and boosting tissue maintenance and repair.

GW0742 but not VEGF-A-induced EC tubulogenesis is dependent on PPARβ/δ. We next confirmed that agonist-activated PPARβ/δ stimulates EC dynamic behaviour in vitro. As GW0742 exerts some of its effects independently of PPARβ/δ 16 , the requirement for PPARβ/δ activation was confirmed using GSK0660, a PPARβ/δ antagonist. As shown in Fig. 2, incubation with GW0742 (100 nM) did not influence HUVEC migration, but significantly increased tubulogenesis in the tube formation assay. This response was abolished in the presence of a maximally effective concentration of GSK0660 (1 μM) ( Fig. 2b and Supplementary Table S1), suggesting that the pro-tubulogenic effect of GW0742 was mediated through PPARβ/δ. As expected, VEGF-A (25 ng/ml) enhanced both EC migration and tube formation, with the latter being unaffected by treatment with GSK0660 ( Fig. 2 and Supplementary Table S1), indicating that VEGF-A-induced HUVEC motility is not dependent on PPARβ/δ.

VEGF-A and GW0742 exert differential forms of metabolic activation in motile HUVEC. As in
HUVEC monolayers, stimulation of tube formation with VEGF-A (25 ng/ml; 4 h) led to a 9-fold increase in glycolytic rate compared with untreated controls (Fig. 3a). In addition, a significant increase in LDHA and MCT1 gene expression was apparent at the time that mature EC networks were established (16 h) ( Fig. 3d-f). Of note, despite the reduced rate of FAO in cell monolayers, FAO in VEGF-A-treated tube-forming cells was maintained (Fig. 3c), whilst CPT1A and CACT mRNAs were unaffected (Fig. 3g,h). On the other hand, stimulation of tube formation with GW0742 (100 nM) resulted in a significant 2-fold increase in FAO compared to untreated controls (Fig. 3c), whilst glycolytic flux was maintained (Fig. 3a). It is interesting to note that although GW0742 increased FAO, a significant up-regulation of LDHA mRNA expression was seen in established tubes (16 h) (Fig. 3d), while both CACT and CPT1A expression was significantly reduced (Fig. 3g,h). As observed in cell monolayers, glucose oxidation rate was unaffected by VEGF-A or GW0742 treatment (Fig. 3b). To determine if these metabolic changes influenced the level of ATP within dynamic cells, change in relative ATP content was assessed. It was not possible to assess ATP in the small number of cells used in the tube-formation assay, but a significant increase in ATP content was seen in active sub-confluent cells following treatment with VEGF-A but not with GW0742 (Fig. 3i).
The transcriptional response observed in cardiac EC from PPARβ/δ overexpressing mice is similar to that elicited in HUVEC by GW0742. To identify whether PPARβ/δ-induced angiogenesis in vivo is associated with similar changes in EC metabolic phenotype, cardiac ECs from the previously-reported inducible conditional endothelial-specific mouse model of PPARβ/δ overexpression were utilised 17 . In this model, Cre-mediated PPARβ/δ overexpression is induced approximately 3-fold upon treatment with tamoxifen and leads to induction of an angiogenic programme within the heart 17 . RT-qPCR analysis of murine cardiac ECs immediately following their isolation revealed a similar reduction in mRNA expression of genes encoding enzymes involved in both glucose (LDHB) and lipid metabolism (CPT1A and CACT) in cells isolated from mice 1 week following treatment with tamoxifen (33 mg/kg/day), compared with animals treated with vehicle alone (Fig. 3j). These data suggest that in line with the role of PPARβ/δ identified in other tissues [11][12][13] , PPARβ/δ-induced angiogenic activity is associated with a shift in EC metabolic phenotype.
An intact glycolytic network is of greater importance for EC tubulogenesis than mitochondrialderived ATP production. Given the changes to glycolysis and mitochondrial metabolism seen in both GW0742-and VEGF-A-treated cells, we next assessed the importance of glycolytic flux and mitochondrial-derived ATP production for each agonist in promoting tubulogenic behaviour.
First, using the metabolic flux data reported in Figs. 1 and 3, the potential contribution of mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis to ATP production was calculated. In resting HUVEC, assuming that 100% of the 14 CO 2 detected from U-14 C-glucose metabolism arose from the TCA cycle, this would translate to <50% of calculated ATP production (from the three pathways tested) being supplied through mitochondrial OXPHOS (Fig. 4a). However, as studies show that <6% of measured 14 CO 2 metabolised by ECs arises from TCA cycle activity 18 , the contribution of mitochondrial OXPHOS to ATP production is likely to be even lower, with the majority (>70%) instead being supplied through anaerobic glycolysis (Fig. 4a). This is in line with that reported by others 3 . Even when cells were undergoing agonist-induced tubulogenesis, the estimated contribution of mitochondrial-derived ATP remained much lower than that supplied by glycolysis (Fig. 4b).
To directly assess the requirement for a functioning mitochondrial ATP-synthase in fuelling HUVEC dynamic activity, we made use of the ATP synthase inhibitor, oligomycin A. Unexpectedly, treatment with oligomycin A (2 μM; 16 h) led to a significant and robust induction of tube formation, comparable to that induced by the other two agonists (Fig. 4c and Supplementary Table S2). When oligomycin A was combined with VEGF-A, no inhibitory or additive effects were evident. However, when GW0742 and oligomycin A were combined, the number of tubes formed was still significantly greater than in control conditions, but other parameters measured, such as the number of junctions, were not (see Supplementary Table S2). These data indicate that ATP production through mitochondrial ATP synthase is not a fundamental requirement for supporting HUVEC dynamic behaviour in vitro, particularly in the presence of VEGF-A. However, impedance of mitochondrial ATP synthase activity may partially disrupt the actions of GW0742 in terms of connectivity of formed tubes but not overall tube number.
In contrast, when glycolytic flux was suppressed using the 6-phosphofructo-2-kinase/fructose-2,6biphosphatase 3 (PFKFB3) inhibitor, 3PO (15 μM) 3,19 , HUVEC were unable to form capillary-like structures after stimulation with either VEGF-A (25 ng/ml) or GW0742 (100 nM) (see Supplementary Fig. S1), and a complete cytostatic effect was observed in the presence of the PFKFB3 inhibitor. However, HUVEC, in both the tube formation assay and in monolayers, were still capable of reducing MTT, indicating that a degree of mitochondrial function was retained under these conditions. Furthermore, in cell monolayers, exposure to 3PO (15 μM; 16 h) was associated with a 15% reduction in mitochondrial reducing potential (see Supplementary Fig. S1), suggesting that 3PO treatment may lead to an overall reduction in metabolic activity.
As an alternative means of reducing glycolytic flux, HUVEC were co-incubated with 10 mM exogenous L-lactate, a concentration sufficient to reduce glycolytic flux in HUVEC monolayers (see Supplementary Fig. S2). The presence of exogenous L-lactate led to a significant reduction in VEGF-A-induced tube formation but did not   Table S3). Moreover, L-lactate alone induced dynamic tube formation. A reduced lactate concentration in the medium (from 10 to approximately 5 mM) at the end of the assay (16 h), suggests that this positive motile effect was associated with the direct uptake of L-lactate by HUVEC (see Supplementary Fig. S2). Although glucose oxidation was significantly reduced in cells undergoing L-lactate-induced tubulogenesis versus untreated control cells, interestingly the rate of glycolysis was maintained, and a cytostatic effect was still observed in the presence of the glycolysis inhibitor, 3PO (see Supplementary Fig. S2). When LDH was directly inhibited with oxamate (1 mM), a structural analogue of pyruvate, this reduced HUVEC tubulogenesis under basal, VEGF-A, GW0742, and L-lactate treated conditions ( Fig. 4e and Supplementary Fig. S3 & Table S4), suggesting that an active LDH enzyme is essential for supporting HUVEC dynamic behaviour, independent of the stimulus.
Mitochondrial FAO contributes to growth factor-induced EC growth and motility. Although not a major contributor to EC ATP production, a healthy and functional mitochondrial network is vital for efficient  . Mitochondrial ATP synthesis contributes less than glycolysis to HUVEC ATP production and is not essential for HUVEC tubulogenesis. (a) Glycolysis (Gly), compared with glucose oxidation (GO) and FAO, provides most of the estimated ATP under basal contact-inhibited conditions when assuming either 100% or 6% of the 14 CO 2 detected from D-U-14 C-glucose metabolism arises from the TCA cycle. (b) Glycolysis, compared with glucose oxidation and FAO, is the largest contributor to estimated ATP production rate in dynamic HUVEC under basal, VEGF-A (25 ng/ml) and GW0742 (100 nM) treated conditions when assuming 6% of the 14 CO 2 detected from D-U-14 C-glucose metabolism arises from the TCA cycle. (c) Inhibition of mitochondrial ATP synthase with oligomycin A (2 µM) lead to a significant increase in the number of capillarylike tubes formed by HUVEC at 16 h and had no significant effect on VEGF-A (25 ng/ml) or GW0742 (100 nM) induced tubulogenesis. Data are means (±S.E.M) number of branches/field from n = 3. *p < 0.05 **p < 0.01 vs. untreated control; ## p < 0.01; as determined by two-way repeated measures ANOVA followed by Bonferroni's post-comparison test. (d) L-lactate (10 mM) significantly increased the number of capillary-like structures formed by HUVEC at 16 h and significantly reduced VEGF-A (25 ng/ml), but not GW0742 (100 nM)-induced tube formation. Data represent means (±S.E.M) of n = 6; **p < 0.01 vs. untreated control; # p < 0.05 ## p < 0.01; as determined by two-way repeated measures ANOVA followed by Bonferroni's post-comparison test. (e) Direct inhibition of LDH with oxamate (1 mM) lead to significant reduction in capillary-like tube formation by HUVEC at 16 h under basal, VEGF-A (25 ng/ml), GW0742 (100 nM) and L-lactate (10 mM) conditions. Data represent mean (±S.E.M) number of branches/field from n = 5. **p < 0.01 ****p < 0.0001 vs. untreated control; #### p < 0.0001; as determined by two-way repeated measures ANOVA followed by Bonferroni's postcomparison test.
Treatment of confluent HUVEC with VEGF-A (25 ng/ml; 4 h) was not associated with any significant change in mRNA expression of mitochondrial transcription factor A (TFAM), a transcription factor involved in mitochondrial biogenesis, or the mitochondrial fusion protein 2 (MFN2) (see Supplementary Fig. S4). However, VEGF-A treatment did result in a significant increase in relative mitochondrial DNA content (p = 0.02), as measured by qPCR, and a subtle change in mitochondrial morphology and number by visual inspection following incubation with MitoTracker Green (see Supplementary Fig. S4). No change in either TFAM mRNA expression, mitochondrial DNA content, or MitoTracker Green staining was detected following treatment with GW0742 (100 nM; 4 h) (see Supplementary Fig. S4). However, after VEGF-A and GW0742-induced tube formation (16 h), mitochondria appeared more aligned within the tubes and there was an increase in MFN2 mRNA expression (see Supplementary Fig. S4), suggesting a common change in mitochondrial tethering upon HUVEC tubulogenesis.
It has recently been suggested that mitochondrial FAO is optimised to uniquely supply carbon for the synthesis of nucleotides necessary for EC proliferation 5 . As an elevated rate of FAO was observed in GW0742-induced tubulogenic cells (see Fig. 3c), the effect of GW0742 treatment on HUVEC proliferation was assessed.
As expected, VEGF-A (25 ng/ml) significantly enhanced HUVEC proliferation at 24 h, as assessed by BrdU incorporation (Fig. 5a,b) and nuclear counting ( Supplementary Fig. S5), whereas treatment with GW0742 (100 nM) had no effect. To understand the importance of mitochondrial FAO for VEGF-A-induced HUVEC proliferation, siRNA was used to achieve targeted knock-down of CPT1A. Immunofluorescence imaging showed effective silencing of CPT1A was achieved at a siRNA concentration of 10 nM using two independent siRNAs (Fig. 5c). This was also confirmed by RT-qPCR (for siRNA 1 & 2) and western blot (for siRNA 1 www.nature.com/scientificreports www.nature.com/scientificreports/ only) (Supplementary Fig. S5). The increase in HUVEC proliferation in response to VEGF-A (25 ng/ml; 24 h) was still evident in cells transfected with non-coding siRNA (10 nM), as assessed by BrdU incorporation, but was abolished in CPT1A-silenced HUVEC (Fig. 5d and Supplementary Fig. S5), supporting a role for FAO in VEGF-A-induced HUVEC proliferation.
As the PPARβ/δ agonist did not affect HUVEC proliferation, the importance of mitochondrial FAO for HUVEC growth into capillary-like branches was investigated. Although siRNA against CPT1A successfully reduced CPT1A expression, PPARβ/δ mRNA expression was also significantly down-regulated in cells where CPT1A had been silenced (Fig. 5e). This effect was observed following silencing with both CPT1A-targeting siRNAs, suggesting an intricate relationship between CPT1A-governed FAO and PPARβ/δ expression, making it difficult to decipher if any functional effect on GW0742-induced tubulogenesis reflects a reduction in CPT1A activity or the associated reduction in PPARβ/δ expression.
To avoid the problem with CPT1A silencing, we then used a pharmacological approach employing the generic CPT1 inhibitor, etomoxir (ETO). ETO is widely used at concentrations of 40 μM or higher 4 . However, the presence of 40 μM ETO in the tube-formation assay led to significant cell death, as indicated by the detachment of cells from the substratum. In subsequent investigations using ETO at 1 and 10 μM, a varied response by individual HUVEC isolates was observed. In some cases, the presence of ETO at either concentration led to significant cell detachment from the matrix, whilst in other isolates, ETO had no effect on basal tube-formation but reduced the number of tube-like branches formed under both agonist-treated conditions after 16 h (see Supplementary  Fig. S6).
Although it was not possible to reliably confirm a role for FAO in agonist-induced tubulogenesis, these data suggest that disruption of FAO for a prolonged period can significantly disrupt EC growth and proliferation.

GW0742 but not VEGF-A-induced tubulogenesis is dependent on the metabolic co-regulator SIRT1.
PPARβ/δ-mediated effects have been reported to be SIRT1-dependent 22 , and SIRT1 itself has recently been linked to the metabolic regulation of angiogenesis through a mechanism involving deacetylation of the FOXO1 transcription factor [23][24][25] . This prompted us to investigate the role of the SIRT1/FOXO1 axis in facilitating GW0742-induced tube formation.
As indicated in Supplementary Fig. S6, immunofluorescence staining showed that SIRT1 was predominantly located within the nuclear and perinuclear region of HUVEC when cultured as contact-inhibited monolayers. Importantly, SIRT1 maintained its nuclear location upon stimulation to form tube-like structures with either GW0742 (100 nM) or VEGF-A (25 ng/ml). No significant increase in SIRT1 mRNA expression was observed under any treatment condition, either in HUVEC monolayers or following tubulogenesis (see Supplementary  Fig. S7). When SIRT1 activity was inhibited using the highly-selective inhibitor, Ex-527 (1 μM), GW0742-induced tube formation was significantly reduced, whereas basal and VEGF-A-driven tubulogenesis were unaffected ( Fig. 6a and Supplementary Table S5).
To identify whether the SIRT1-dependency of GW0742's effect on tube formation was due to the requirement for SIRT1-mediated regulation of FOXO1, the acetylation status of FOXO1 was investigated. Immunofluorescence microscopy using an antibody specific for acetylated FOXO1 (acFOXO1) showed that the acetylated form of this transcription factor was almost exclusively located within the nuclear compartment of HUVEC and that the degree of acetylation was not influenced by either GW0742 (100 nM; 1 h) or VEGF-A (25 ng/ml; 1 h) ( Fig. 6b and Supplementary Fig. S7). As VEGF-A is known to rapidly (within 1 h post-stimulation) inactivate FOXO1 through Akt-mediated phosphorylation 26,27 , the phosphorylation status of FOXO1 (pFOXO1) was measured in parallel. As expected, treatment of confluent HUVEC with VEGF-A (25 ng/ml; 1 h) led to an increase in FOXO1 phosphorylation, as assessed by western blotting, while incubation with GW0742 (100 nM; 1 h) did not affect FOXO1 phosphorylation status (Fig. 6c,d).
Together these data show that SIRT1 is predominantly located within the nucleus of endothelial cells and that whilst VEGF-A leads to FOXO1 inactivation and SIRT1-independent HUVEC tubulogenesis, GW0742-induced HUVEC tubulogenesis is a SIRT1-dependent process but independent of any significant change in FOXO1 status.

Discussion
The importance of metabolism for regulating angiogenic behaviour is increasingly recognised 3,5 but knowledge of how ECs adapt their metabolism in response to extrinsic signals is still lacking. Although a pro-angiogenic role for the PPARβ/δ nuclear receptor has been demonstrated 7,10,17 , whether this effect is mediated through alterations in EC metabolism has not been thoroughly investigated. We therefore aimed to compare the functional and metabolic responses of HUVEC treated with the pharmacological PPARβ/δ agonist, GW0742, with that of VEGF-A which has well-established proliferative, migratory and tubulogenic effects 28 .
The results presented here suggest that unlike VEGF-A, treatment of HUVEC with a selective concentration of GW0742 (100 nM) 15 is not sufficient to induce short-term proliferation or migration. In line with this, it has been reported that GW0742 fails to induce proliferation of human retinal microvascular ECs at 24 h 10 , and similar results have also been described with the alternative PPARβ/δ agonist, GW501516 29,30 . This contrasts with findings reported by others demonstrating pro-proliferative and pro-migratory effects of PPARβ/δ agonists on ECs 7,31,32 . These discrepancies may reflect the time-point analysed, as the pro-proliferative effects were principally observed following long-term treatment (3-14 days) as opposed to the 24 h time point analysed here.
The contrasting functional responses of HUVEC to GW0742 and VEGF-A stimulation may be explained in part through the differential metabolic response induced by these two agonists. VEGF-A activates multiple signalling pathways that can lead to an increased rate of glycolysis and induction of mitochondrial biogenesis in preparation for cell proliferation 3,20 , a response also observed in the present study in HUVEC monolayers. Increased glycolytic flux can support migratory behaviour as many glycolytic enzymes are located alongside cytoskeletal proteins, providing hotspots of localised ATP production 3 . A high level of glycolysis also supports proliferation, as flux through glycolysis and its associated side-pathways provides the carbon necessary for the generation of new biomass 33,34 .
In contrast, treatment of HUVEC monolayers with GW0742 caused a reduction in glycolysis, FAO and oxygen consumption, indicating that PPARβ/δ may instead facilitate a form of metabolic quiescence. Importantly, this response was not associated with a compromise in overall mitochondrial activity, as no changes in mitochondrial DNA content, MitoTracker staining or MTT reducing capacity were observed, and cellular ATP content remained stable. This phenotype could, in part, contribute to the known anti-inflammatory and cyto-protective effects of PPARβ/δ agonists within the vasculature [35][36][37][38] , as a less active metabolism could be a mechanism by which ECs afford further protection from unnecessary oxidative stress arising from mitochondrial ROS production 39 . Furthermore, lowered EC metabolic rate could optimise nutrient transfer through the endothelium, thereby facilitating the delivery of nutrients to underlying tissues, as has been indicated in the heart 40 . It is tempting to speculate that these metabolic effects may contribute to the perceived benefits exerted by PPARβ/δ agonists on reperfusion-induced injury [41][42][43] . Interestingly, the metabolic response following GW0742 treatment demonstrated here is similar to that described for other vascular protective stimuli 25,44 , suggesting that this may be a common cyto-protective mechanism.
Whilst migration and proliferation were unaffected by GW0742 treatment, both GW0742 and VEGF-A were able to exert a tubulogenic effect. Similar pro-angiogenic responses to PPARβ/δ activation have been reported previously [7][8][9][10]17 . Furthermore, we now show that this tubulogenic effect is dependent on the deacetylase enzyme and metabolic co-regulator, SIRT1. SIRT1 signalling has previously been linked with regulating mitochondrial FAO 45 , as well as co-ordinating angiogenic activity, with SIRT1-silenced HUVEC showing a reduction in PDGF-β expression 23,24 , a key mediator in the recruitment of mural cells necessary for vessel stabilisation 46 . Given the lack of effect of the PPARβ/δ agonist on EC migration and proliferation, it seems reasonable to propose that PPARβ/δ may co-operate alongside SIRT1 within the nuclear compartment to regulate neo-vessel maturation/stabilisation. It is possible that several SIRT1 target proteins are involved in facilitating the tubulogenic response 23,24,47 . One such target is the FOXO1 transcription factor which also plays a vital role in the regulation of EC metabolism and in the maturation/stabilisation phase of angiogenesis 25 . However, the absence of any significant change in www.nature.com/scientificreports www.nature.com/scientificreports/ acetylation or phosphorylation with GW0742 treatment suggests that FOXO1 is unlikely to be a major target in this instance. Given that only a short time-point was investigated, involvement of FOXO1 at a later time-point cannot be ruled out.
Vessel maturation may also be supported through alterations in mitochondrial FAO. FAO has been shown to be required for the establishment of stable networks on Matrigel TM through facilitating EC-EC contact and regulating permeability 4 . In keeping with this, SIRT1-dependent GW0742-induced tubulogenesis was associated with an increased rate of FAO, a response not seen with exposure to VEGF-A. Unexpectedly, however, by 16 h of tubulogenesis in the presence of GW0742, both CACT and CPT1A genes had become down-regulated. This profile was also evident in cardiac ECs isolated from mice with selective endothelial PPARβ/δ overexpression, a model characterised by enhanced angiogenesis within the heart 17 . Although we cannot exclude that mouse cardiac ECs may have a slightly different metabolic response to that of HUVEC, the data for both cell types are in close agreement with each other. As such, this down-regulation of gene expression at the latter stages of both GW0742-induced HUVEC tubulogenesis in vitro and PPARβ/δ-induced angiogenesis in vivo may indicate a regulatory feedback loop designed to prevent a persistently elevated rate of FAO that could lead to a disturbance in local metabolic homeostasis. Indeed, rather than the physiological cardiac hypertrophy observed with the PPARβ/δ agonist, endothelial-specific PPARβ/δ overexpressing mice develop a pathological cardiac hypertrophy that was suggested to be a consequence of an altered balance of PPARβ/δ activity between the vascular and muscular compartments 8,17 .
Although an induction of FAO by PPARβ/δ activation has been established, its functional role remains unclear. Despite respiration in ECs being highly coupled with ATP synthesis 48 , increasing FAO to fuel mitochondrial-derived ATP does not appear to be a primary factor, as incubation with oligomycin, an inhibitor of mitochondrial ATP synthase, did not significantly impair the ability of HUVEC to form tube-like structures. This is in line with the fact that ECs principally obtain their ATP through glycolysis 3 . Moreover, an increase in tube-formation at 16 h of oligomycin treatment was evident, likely because of a compensatory increase in glycolytic flux that is known to occur following ATP synthase inhibition 48 . When coupled with the basal level of stimulation provided by the matrix, this adaptation may be enough to induce HUVEC tubulogenesis in vitro. Reports also present the possibility of oligomycin-induced VEGF production 49 . However, this effect was described in a monocytic cell line and further investigation would be required to determine whether similar mechanisms are operative in ECs. Beyond ATP production, fatty acids offer a rich source of carbon beneficial for generating biomass. ECs appear to be one of only a few cell types to utilise FAO for supplying carbon to support nucleotide synthesis vital for proliferation 5 . Although this role for FAO has yet to be confirmed by others, in support of such a concept, the VEGF-A-induced proliferation reported in the current study was completely abolished in CPT1A-silenced HUVEC. However, it is unlikely that the increase in FAO following GW0742 treatment is used to fuel proliferation, as proliferative activity was not affected by GW0742 treatment (as discussed above), and would not be expected to play a significant role over the time-frame of the tube-formation assay (16 h).
Understanding the role of FAO in facilitating GW0742-induced tubulogenesis was made challenging by the tight relationship that exists between the PPARβ/δ signalling pathway and FAO. Variable responses by HUVEC to the generic CPT inhibitor, ETO, made the pharmacological approach unreliable. However, when attempts were made to silence CPT1A, this led to a concomitant reduction in PPARβ/δ gene expression. Despite attempts to measure PPARβ/δ protein, by both western blotting and immunofluorescence analysis, the lack of reliable, commercially available PPARβ/δ-selective antibodies 50 meant that protein levels could not be reliably assessed. Despite not being able to perform tube-formation assays in CPT1A-silenced cells, it is tempting to suggest that such a reciprocal regulatory relationship between PPARβ/δ and CPT1A is an integral part of GW0742-induced tubulogenesis, where CPT1A-driven FAO influences PPARβ/δ transcriptional activity and possibly PPARβ/δ expression itself. Indeed, a similar association has been described in lymphatic ECs for the transcription factor PROX1 51 . CPT1A is a PROX1 target and CPT1A-driven FAO provides acetyl-CoA that can be utilised for acetylation-mediated epigenetic modifications that facilitate the transcriptional activity of PROX1 and its target genes 51 . Any such molecular interaction with PPARβ/δ, however, requires further investigation.
It is important to note that the increase in FAO by GW0742 is not at the expense of glucose metabolism, as glucose oxidation and glycolysis were maintained, the latter even slightly increasing, thereby contrasting with the response in EC monolayers. The advantage that glycolysis holds for facilitating motile behaviour has been outlined above and its importance is highlighted by the cytostatic effect observed following inhibition of glycolysis with the PFKFB3 inhibitor, 3PO. This effect was independent of the stimulus used and is identical to the response observed by Schoors et al. 19 . Moreover, the reliance in particular on anaerobic glycolysis (even in the presence of sufficient oxygen) to support HUVEC growth and motility was indicated by the up-regulation of LDHA mRNA in both VEGF-A-and GW0742-stimulated cells and by the global impairment in tubulogenesis in the presence of the LDH inhibitor, oxamate. This requirement for LDH-facilitated anaerobic glycolysis has previously been described in pulmonary microvascular ECs in vitro and in vivo 52,53 . Although a baseline level of LDH-facilitated anaerobic glycolysis is essential, the extent to which it requires up-regulating to support tubulogenic activity is agonist-dependent, as maximal tubulogenesis induced by VEGF-A, which was also the largest inducer of glycolytic flux, but not GW0742, was impaired when anaerobic glycolysis was maintained towards baseline levels through supplementing the culture medium with L-lactate. It is also worth noting that L-lactate alone exerted a pro-tubulogenic response, an effect also demonstrated by others 54 .
Together, these findings expand our knowledge of the role of PPARβ/δ within the endothelium by identifying a context-dependent regulation of EC metabolism in vitro by GW0742. In contrast to the glycolytic activation observed in VEGF-A-stimulated EC monolayers and dynamic cells (Fig. 7a), GW0742-activated PPARβ/δ promotes a degree of metabolic quiescence in cell monolayers, but metabolic activation, in the form of elevated mitochondrial FAO, in tubulogenic cells (Fig. 7b). These actions are dependent on the metabolic co-regulator SIRT1. Further studies are now needed to decipher the specific interaction between these two metabolic regulators, as Scientific RepoRtS | (2020) 10:7849 | https://doi.org/10.1038/s41598-020-63900-0 www.nature.com/scientificreports www.nature.com/scientificreports/ well as the functional role of GW0742-induced FAO within the angiogenic process, with a view to exploit these findings for therapeutic intervention.

Methods
The data sets generated during the current study are available from the corresponding author on reasonable request. cell isolation and culture. Human umbilical cord collection (obtained with informed written consent) and use of human endothelial cells conformed to the principles outlined in the Declaration of Helsinki and is approved by the NHS Health Research Authority East of England-Cambridge South Research Ethics Committee (REC reference 16/EE/0396). All experiments were performed in accordance with relevant guidelines and regulations. Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as described previously and were used between passage 2 and 4 55 . Cells were cultured on 1% gelatin and maintained in M199 growth medium (M199 containing endothelial cell growth factor (20 μg/ml) and 20% (v/v) FBS). Experiments were performed in HEPES-buffered M199 containing 5mM L-glucose, 5mM L-glutamine, 1% FBS and 1% penicillin/streptomycin. Endothelial cells were isolated from mouse hearts and gene expression analysed by reverse transcription quantitative PCR (RT-qPCR). All animal work was conducted according to national and international guidelines and was approved by the local ethics committee (Comité Institutionnel d'Éthique Pour l' Animal de Laboratoire Azur, agreement number: PEA-NCE/2013/106). Age-and sex-matched tamoxifen-inducible Cre-mediated vascular-specific PPARβ/δ overexpressing animals (Tie2 CreERT2;PPARβ/δ) were injected for one week intraperitoneally either with sunflower oil (vehicle) or tamoxifen dissolved in sunflower oil (33 mg/kg per day), as previously described 17 . Tie2-CreERT2 animals injected with tamoxifen served as an additional control. Following terminal anaesthesia, hearts were excised, and cardiac endothelial cells were isolated by magnetic-activated cell sorting (MACS) using anti-CD31 beads (Miltenyi Biotec) prior to analysis by RT-qPCR. tube formation assay. Tubulogenesis was assessed using the thin-layer angiogenesis (TLA) assay described previously 55 . Briefly, 5 μl/cm 2 of Geltrex TM basement matrix (ThermoFisher Scientific; UK) was spread evenly on to wells of 24-or 96-well plates as appropriate. HUVEC were seeded (25,000 cells/cm 2 ) in technical duplicate (24-well plate) or sextuplet (96-well plate) and incubated for 16 h in the presence or absence of compounds of interest. Phase-contrast images (24-well plate: 4 images/well; 96-well plate: 1 image/well) were acquired using a Leica DMIRB inverted microscope (10x objective (NA = 0.25)) controlled through Axiovision software version (a) Treatment with VEGF-A is characterised by a significantly high rate of glycolysis which provides ATP for migration and activation of biosynthetic side-pathways for proliferation. At the same time, mitochondrial fatty acid oxidation (FAO) is maintained, which may further support proliferation through acting as an additional source of carbon. Key metabolites can also influence angiogenesis-related signalling pathways while Akt, through inhibitory phosphorylation, can inactivate the anti-proliferative transcription factor, FOXO1. (b) GW0742-induced tubulogenesis is SIRT1-dependent and characterised by a significant increase in mitochondrial FAO in addition to a moderate increase in glycolysis. Elevated acetyl-CoA levels may act as a substrate for post-translational protein acetylation (indicated in the figure by ) which could facilitate PPARβ/δ transcriptional activity and influence angiogenesis-related signalling pathways. FAO may also contribute to the regulation of EC permeability and thus may have a role in neovessel maturation/stabilisation. Thickness of arrows indicate increased flux while broken arrows indicate pathways that remain to be established.

Mitochondrial activity.
Overall mitochondrial activity was assessed by 3-(4,5 dimethylthiazol-2-yl)-2 ,5-diphenyltetrazolium bromide (MTT) reduction assay. After treatment, cells were incubated for 2 h (37 °C/5% CO 2 ) with MTT solution (1 mg/ml in PBS) and the resultant formazan product was solubilised using solubilisation solution (DMSO; 37 °C; 20 min). Solubilisation solution was collected and transferred to a clear 96-well plate (50 µl/well in technical triplicate) prior to measurement of absorbance at 550 nm. Readings were corrected for background and results are expressed as a percentage of control.
Statistical analysis. All data are expressed as means ± standard error of means (S.E.M) and unless stated otherwise, are from at least 3 independent experiments performed on separate EC isolates. Data analysis was performed using GraphPad Prism 6 software. Student's t-test was employed for establishing significant differences between two groups. For the comparison of three or more groups analysis of variance (ANOVA) was applied followed by Bonferroni or Tukey post-hoc analysis as appropriate. In all cases, p < 0.05 was used to indicate statistical significance.