We investigated whether the release of proinflammatory cytokines or the accumulation of Kyn affects the viability of HCEC. Cell death was determined by detection of 7-aminoactinomycin (7-AAD, a marker for the early phase of apoptosis) and Annexin V (a marker of cell death) upon treating HCEC with IFN-γ or TNF-α (
Fig. 5, first and second rows), or by adding various concentrations of Kyn (100, 200, 400, 800, and 1600 μM;
Fig. 6, graph, third row). The results showed a statistically not significant increase of 7-AAD after incubation with IFN-γ (
P = 0.13, compared to untreated HCEC,
Fig. 5, first row) and a stronger, statistically significant increase after TNF-α treatment (
P = 0.02, compared to untreated HCEC,
Fig. 5, first row). For Annexin V, similarly, an increase was observed after incubation with IFN-γ (
P = 0.13, compared to untreated HCEC,
Fig. 6, second row), whereas treatment with TNF-α caused stronger, statistically significant increase (
P = 0.03, compared to untreated HCEC,
Fig. 5, second row). In experiments using Kyn, apoptosis occurred only at concentrations of >800 μM (
P ≤ 0.01, compared to treatment without Kyn,
Fig. 6, third row). These concentrations clearly exceed those expected in vivo.