Extended Data Figure 10: Pharmacological inhibition of FAO results in defects in angiogenesis.

a, b, Quantitation of lymphatic vessel end-point density (number of vessel extremities per corneal area; a) and lymphatic vessel length density (cumulative length of the lymphatic vessel network; b), both parameters per total corneal area, in wild-type mice subjected to corneal cauterization-induced injury treated daily with vehicle (ctrl) or etomoxir in Fig. 5e–k (n = 5 animals per group). c, Representative images of CD31⁺ blood vessel outgrowth 9 days after corneal cauterization-induced injury in adult wild-type mice treated daily with vehicle (ctrl) or etomoxir. Scale bars, 250 μm. d–g, Quantitation of percentage blood vessel outgrowth area relative to total area (d), branch point density (e), end-point density (f) and length density (g) in wild-type mice subjected to corneal cauterization-induced injury treated daily with vehicle (ctrl) or etomoxir from the corneal cauterization experiments in Fig. 5e–k (n = 5 animals per group). h, Measurement of plasma acetate concentrations in mice treated with PBS (ctrl) or 400 μl of 0.5 M acetate (ac) for 9 days (n = 6 mice per group). i, j, Quantitation of lymphatic vessel end-point density (number of vessel extremities per corneal area; i) and lymphatic vessel length density (cumulative length of the lymphatic vessel network) (j), both parameters per total corneal area, in wild-type mice subjected to corneal cauterization-induced injury treated daily with vehicle (ctrl), acetate only (ac), etomoxir only (eto) or etomoxir plus acetate (eto+ac) from the corneal cauterization experiments in Fig. 6f–j (n = 10 animals per group). k, Traditional and adapted model of the mechanism of PROX1-induced lymphatic formation. Traditional model: PROX1 binds to the promoter region of lymphangiogenic genes, including VEGFR3, enhancing their transcription. Adapted model: by binding to the CPT1A promoter, PROX1 induces CPT1A expression, enhancing the production of FAO-derived acetyl-CoA. In a dual manner, FAO (red) thereby regulates: (1) LEC proliferation through providing acetyl-CoA, which helps to sustain the Krebs cycle and deoxyribonucleotide (dNTP) synthesis in conjunction with an anaplerotic substrate during lymphangiogenesis (purple); and (2) lymphangiogenic gene expression through supplying acetyl-CoA (AcCoA) used for histone acetylation (Ac) at lymphangiogenic genes, thereby modulating LEC differentiation, proliferation and migration (green). PROX1 promotes histone acetylation preferentially at lymphatic genes by enriching p300 at PROX1-binding sites, and by supplying more acetyl-CoA (through FAO induction). Thus, in the adapted model, PROX1 still functions as a transcription factor (as proposed in the traditional model), but its role is extended by using FAO metabolism to enhance its own transcriptional activity. Mean ± s.e.m.; (n = 5 animals per group from 1 experiment in a, b, d–g), (n = 6 animals per group from 1 experiment in h), (n = 6 animals per group from 2 experiments in i, j). Statistical test: ANOVA and Bonferroni post-hoc test were used in multiple group comparisons. t-test was used for comparison of two groups. *P < 0.05.