The Canonical Wnt Pathway in the Pathogenesis of Cardiac Allograft Vasculopathy.

PURPOSE: The canonical wnt pathway with its central signaling molecule β-catenin is important for tissue development and homeostasis and its dysregulation leads to many diseases including pathologies of the cardiovascular system. Inhibition of the wnt pathway has the potential to protect against cardiac allograft vasculopathy (CAV), because it reduces the proliferation of smooth muscle cells (SMCs), which is a main pathomechanism of CAV and causes neointima formation and vessel occlusion.

METHODS: For in vitro experiments human (hu) and murine (mu) aortic SMCs were grown in the presence of increasing concentrations of wnt inhibitors, such as XAV-939, ICG-001 or PKF118-310. Cell proliferation was measured as the extent of PrestoBlue Cell Viability Reagent metabolism and expression of representative wnt target genes was assessed by Taqman PCR. Additionally, the wnt inhibitors were used in the mouse model of aortic transplantation (n=8 mice/group) and the extent of neointima formation as an indicator for CAV was analyzed. We also measured the expression of β-catenin (immunofluorescence staining), wnt target genes (Taqman PCR) and several inflammatory cytokines (Taqman PCR) in the transplant.

RESULTS: In vitro experiments revealed dose-dependent proliferation inhibition in huSMCs and muSMCs after treatment with each wnt inhibitor. PKF118-310 induced cell death in the highest concentrations tested. While XAV-939 had only minor influence on the expression of the wnt target genes cyclin D1, axin2 and p21, ICG-001 and PKF118-310 induced the expression of p21 and cyclin D1 and reduced the expression of axin2. Interestingly, in vivo experiments showed no effect of XAV-939 and ICG-001 on neointima formation, whereas neointimal amounts of β-catenin were significantly reduced. ICG-001 also reduced the expression of inflammatory cytokines (INFγ, TNFα and IL-6) and of p21, but did not influence cyclin D1 expression. XAV-939 had no relevant influence on cytokine expression. Results for PKF118-310 and wnt target gene expression in transplants after XAV-939 treatment are currently assessed.

CONCLUSION: Strong anti-proliferative effects were found in vitro for all three wnt inhibitors, but this did not lead to reduced CAV development in a mouse model of vessel transplantation. Effects on SMCs themselves after ICG-001 and PKF118-310 seem indeed mediated via regulation of wnt target genes.