Figure 4.

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 ¹⁴CO₂ detected from D-U-¹⁴C-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 ¹⁴CO₂ detected from D-U-¹⁴C-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 capillary-like 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 post-comparison test.