Treatment with XAV-939 prevents in vitro calcification of human valvular interstitial cells

Abstract

The development of a substance or inhibitor-based treatment strategy for the prevention of aortic valve stenosis is a challenge and a main focus of medical research in this area. One strategy may be to use the tankyrase inhibitor XAV-939, which leads to Axin stabilisation and subsequent destruction of the β-catenin complex and dephosphorylation of β-catenin. The dephosphorylated active form of β-catenin (non-phospho-β-catenin) then promotes nuclear transcription that leads to osteogenesis. The aims of the present study were to develop an experimental system for inducing in vitro calcification of human aortic valvular interstitial cells (VICs) to investigate the potential anti-calcific effect of XAV-939 and to analyse expression of the Wnt signalling proteins and Sox9, a chondrogenesis regulator, in this model. Calcification of human VIC cultures was induced by cultivation in an osteogenic medium and the effect of co-incubation with 1μM XAV-939 was monitored. Calcification was quantified when mineral deposits were visible in culture and was histologically verified by von Kossa or Alizarin red staining and by IR-spectroscopy. Protein expression of alkaline phosphatase, Axin, β-catenin and Sox9 were quantified by western blotting. In 58% of the VIC preparations, calcification was induced in an osteogenic culture medium and was accompanied by upregulation of alkaline phosphatase. The calcification induction was prevented by the XAV-939 co-treatment and the alkaline phosphatase upregulation was suppressed. As expected, Axin was upregulated, but the levels of active non-phospho-β-catenin were also enhanced. Sox9 was induced during XAV-939 treatment but apparently not as a result of downregulation of β-catenin signalling. XAV-939 was therefore able to prevent calcification of human VIC cultures, and XAV-939 treatment was accompanied by upregulation of active non-phospho-β-catenin. Although XAV-939 does not downregulate active β-catenin, treatment with XAV-939 results in Sox9 upregulation that may prevent the calcification process.

RETRACTED: Overexpression of microRNA-203 Suppresses Proliferation, Invasion, and Migration while Accelerating Apoptosis of CSCC Cell Line SCL-1

“…Tankyrase inhibitor, XAV-939 (MedChemExpress, NJ, USA), has been reported to induce Axin stabilization and to cause subsequent destruction of the β-catenin complex and dephosphorylation of β-catenin. 40 Prior to transfection, cells at the logarithmic growth phase were seeded in a 6-well plate. When density reached 80%–90%, the cells underwent centrifugation at 179 × g for 5 min and were transferred into serum-free Roswell Park Memorial Institute (RPMI) 1640 medium for culture.…”

Molecular Spectroscopic Imaging Offers a Systematic Assessment of Pathological Aortic Valve and Prosthesis Tissue in Biomineralization

“…Various aspects of AV research can be investigated using FT-IR spectroscopy to cover basic science and materials research but also in vitro substance testing analyses [14,[19][20][21][22][23][24]35,48]. Pathological human AV tissue niches with different environmental compositions can exhibit diverse types of apatite and precursors as well as changes in ECM matrix composition and conformation [49].…”

“…The differentiation of these cells to a myofibroblast but also osteogenic type of cells contribute to AV sclerosis and is focus of AV research [61]. Validation of the exact mineral composition in the in vitro setups is an important aspect to reflect their applicability, for example, for substance testing [48,64,65].…”

miR‐122 and the WNT/β‐catenin pathway inhibit effects of both interleukin‐1β and tumor necrosis factor‐α in articular chondrocytes in vitro

“…XAV is a tankyrase 1 and 2 inhibitor that works by stabilizing AXIN2 in the cytosol, while PKF selectively inhibits β-catenin from complexing with TCF-4 in the nucleus. 26 Concentrations for XAV 27,28 and PKF 29 were chosen based on previously published studies. rArCs were treated at 80% confluence with 0, 0.5, or 1 μM of XAV or 0, 0.1, 0.5, or 1 of PKF, or with or without 10 ng/ml of IL-1β or 10 ng/ml TNF-α for 24 h and then harvested as described above.…”