Dichotomy in Hedgehog signaling between human healthy vessel and atherosclerotic plaques

Abstract

The major cause for plaque instability in atherosclerotic disease is neoangiogenic revascularization, but the factors controlling this process remain only partly understood. Hedgehog (HH) is a morphogen with important functions in revascularization, but its function in human healthy vessel biology as well as in atherosclerotic plaques has not been well investigated. Hence, we determined the status of HH pathway activity both in healthy vessels and atherosclerotic plaques. A series of 10 healthy organ donor-derived human vessels, 17 coronary atherosclerotic plaques and 24 atherosclerotic carotid plaques were investigated for HH pathway activity. We show that a healthy vessel is characterized by a high level of HH pathway activity but that atherosclerotic plaques are devoid of HH signaling despite the presence of HH ligand in these pathological structures. Thus, a dichotomy between healthy vessels and atherosclerotic plaques with respect to the activation status of the HH pathway exists, and it is tempting to suggest that downregulation of HH signaling contributes to long-term plaque stability.

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.

Figure 2

Abundant presence of HH ligands in atherosclerotic plaques. (A) Coronary plaques were collected from 17 patients by catheterization, and protein levels of HH ligands were analyzed by Western blot. (B) Carotid plaques were collected by endarterectomy and analyzed for the levels of HH protein using Western blot. Immature forms (as deduced from apparent molecular weight) are abundantly present in plaques. Carotid plaques also contain abundant HH. Thus, atherosclerotic plaques are characterized by the presence of HH ligands.

Figure 3

Absence of response to HH ligands in atherosclerotic plaques. (A) Atherosclerotic plaques were collected from 24 patients undergoing carotid endarterectomy for symptomatic high-grade stenosis of the internal carotid artery. Renal arteries from four family kidney transplantation donors were used as healthy control vessels. HH ligand expression and pathway activity was assessed by quantitative reverse transcriptase PCR. Expression of HH pathway targets GLI1 (B) and PTCH1 (C), as determined by quantitative reverse transcriptase PCR, was correlated with the expression level of HH ligands in atherosclerotic plaques. (D) The low levels or absence of HH target gene products were confirmed by Western blotting analysis in the same 24 subjects. (E) Radioimmunoassay analysis of three major plasma lipoproteins reveals that LDL contains the highest concentrations and VLDL the lowest levels of the endogenous HH signaling antagonist 25-OH vitamin D3 (25-OHvitD₃).