Figure 2. IFN-γ accelerates tryptophan degradation to kynurenine in HCAEC.

A, Targeted LC-MS analysis of 144 common intracellular metabolites. Log₂ of fold changes in intracellular metabolites extracted from HCAEC following 24 h of IFN-γ at 50 ng/mL are depicted on the x-axis with their p values on the y-axis. Mean from 3 independent experiments.

B-C, Intracellular tryptophan (B) and kynurenine (C) in HCAEC measured by LC-MS following 1, 6, 24 h of IFN-γ treatment. Mean ± SEM from 9 biological replicates from 3 independent experiments.

D, IDO1 mRNA expression fold change in HCAEC following the same IFN-γ treatment. Mean ± SEM from 3 independent experiments.

E, IDO1 protein expression changes in HCAEC following the same IFN-γ treatment. Representative Western blot.

F, Intracellular kynurenine/tryptophan ratio over time indicating increased indoleamine 2,3-dioxygenase 1 (IDO1) activity. Mean ± SEM from 9 biological replicates from 3 independent experiments.

G-H, Adhesion molecules, ICAM (G) and VCAM (H), mRNA expression fold changes in HCAEC measured by real-time PCR following 24 h incubation in control Dulbecco’s Modified Eagle Medium (DMEM) (Ctrl) or DMEM deficient in tryptophan (TRP) (−TRP) with (black triangles with hatched bars) or without (red squares) TRP supplementation (−TRP + TRP Supp). Mean ± SEM from 3 independent experiments.

I, Percentages of wound closure of monolayer re-endothelialization following 24 h incubation in the above medium calculated by the following formula = [(open area at 0 h) - (open area at 48 h)]/(open area at 0 h) × 100. Mean ± SEM from 4 independent experiments.

Statistical significance was determined by two-way ANOVA with post hoc Tukey’s multiple comparisons test (B-C, F), Welch’s t-test (D), or one-way ANOVA with post hoc Bonferroni’s pairwise comparisons test (G-I).

* p < 0.05, ** p < 0.01, *** < 0.001, **** p < 0.0001

ICAM: intercellular adhesion molecule; VCAM: vascular cell adhesion molecule.