Influence of β-Catenin on the proliferation of vascular smooth muscle cells and the development of cardiac allograft vasculopathy in mice
Christian Heim1, Annika Kuckhahn1, Martina Ramsperger-Gleixner1, Jörg W Distler2, Bernd M Spriewald2, Michael Weyand1, Stephan M Ensminger3.
1Cardiac Surgery, University of Erlangen-Nürnberg, Erlangen, Germany; 2Medicine, University of Erlangen-Nürnberg, Erlangen, Germany; 3Cardiac Surgery, University of Lübeck, Lübeck, Germany
Objectives: β-Catenin is the central signaling molecule in the canonical wnt pathway. Blocking its actions is associated with anti-proliferative effects on different cell lines including vascular smooth muscle cells (VSMCs), which play amajor role in the development of cardiac allograft vasculopathy (CAV) after heart transplantation. Aim of this study was to analyse the inhibition of β-Catenin as potential target to prevent CAV.
Methods: VSMCs from mouse (muSMCs) and human (huSMCs) arteries were cultivated with increasing concentrations of the β-Catenin signaling inhibitors XAV-939, ICG-001 or PKF118-310. The reduced proliferation ability of muSMCs and huSMCs was detected using PrestoBlue Cell Viability Reagent. To verify β-Catenin inhibition as the mechanism of action, expression of its target genes Axin2, Cyclin D1 and p21 was analyzed. Additionally, the inhibitors were used in vivo to treat mice after aortic transplantation to evaluate the effect on reducing CAV by measuring neointimal thickness (n=8 / group). Additionally, the amount of neointimal β-Catenin was examined by immunofluorescence staining and expression of several inflammatory cytokines was analyzed by Taqman PCR.
Results: All β-Catenin signaling inhibitors showed a dose-dependent proliferation inhibition of cultivated muSMCs and huSMCs. PKF118-310 led to cell death in the highest concentrations used. In vivo though, no relevant reduction in CAV was detectable after treatment with XAV-939 or ICG-001. Respective experiments with PKF118-310 are conducted at the moment. Interestingly, neointimal percentages of β-Catenin were reduced after XAV-939 and ICG-001. ICG-001 treatment also led to reduced expression of INFγ, TNFα and IL-6 while this could not be found after XAV-939 application. β-Catenin target gene expression is currently analyzed.
Conclusion: In vitro, strong anti-proliferative effects of the β-Catenin inhibitors were detected on cultured VSMC, whereas no relevant inhibition of CAV development in a mouse model could be seen. We would speculate at this stage, that this is attributable to the complexity of the wnt/ β-Catenin pathway on the one hand as well as that of CAV development on the other hand.
Deutsche Stiftung für Herzforschung .
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