Figure 1

High activity of the HH pathway is characteristic of healthy human vessels. (A) Schematic depiction of HH signal transduction in the absence (left panel) and presence of ligand (right panel). (B) Comparison of GAPDH and RFLP0 expression levels in healthy vessels and plaques. HH pathway target genes GLI1 (C, D) and PTCH1 (E, F) transcript levels were correlated with the expression levels of IHH (C, E) and DHH (D, F) in renal arteries from four healthy organ donors. (G) HH pathway activity in renal arteries from 10 healthy organ donors (sequentially numbered) was assessed using Western blot for the target genes. (H) Human coronary endothelial cells were cultured according to routine procedures for 24 h with either vehicle control or recombinant ShhN (1 µg/mL). Cell viability was evaluated by MTT reduction. Data are means ± standard error of the mean (n = 3) (p < 0.05). Because expression levels of the routinely used GAPDH housekeeping gene may be changed depending on HH pathway activity, we first compared GAPDH levels with RPLP0 levels, since the latter gene was suggested to be a better housekeeping gene when studying HH pathway activity (27). As shown in (B), GAPDH and RPLP0 levels correlate well in all samples used for this study, and we thus considered GAPDH an appropriate housekeeping gene.