Osteopontin Does not Inhibited Aortic Valve Fibrosis and Calcification in Three-Dimensional Ovine Aortic Valve Model

Objectives: Calcific Aortic Valve Disease is characterized by aortic valve fibrosis followed calcification resulting in left ventricular outflow obstruction. Osteopontin (OPN), a multifunctional phosphoprotein, is described to be involved in the inhibition of biomineralization of dystrophic and ectopic sites, including aortic valve tissue, depending on the phosphorylation status of OPN. Here, we investigated whether the described effect of OPN on smooth muscle cells can be transferred to ovine aortic valve cell culture models.

Methods: Ovine aortic valve explants (AVE) were cultivated statically under pro-fibrotic and pro-calcific conditions (5 ng/mL TGF-β, 10 mM β-glycerolphosphate/1.5 mM calcium chloride) for 14 days in presence or absence of 100 ng/mL bovine milk OPN. Phosphorylation status of OPN was analyzed by immunoblot using phospho-specific antibodies. Gene expression in AVE was measured by qPCR, morphological changes were analyzed by hematoxylin-eosin staining. Calcification was assessed by von Kossa staining and alkaline phosphatase assay kit

Result: Pro-fibrotic and pro-calcific cultivation induced morphological changes in AVE. Beside shrinkage and thickening of AVE, even cell distribution was disturbed with enhanced cell proliferation marginally and cell-free regions within the AVE. Simultaneously, these conditions significantly enhanced gene expression of fibrosis markers collagen type I (10.4-fold, n = 9, p < 0.05) and α-smooth muscle actin (12.4-fold, n = 9, p < 0.05) and the process-controlling Runt-related transcription factor (RUNX)2 (2.53-fold, n = 9). In line with these results, collagen protein level (4.8-fold, n = 3) and alkaline phosphatase (1.3-fold, n = 8, p < 0.001) activity in the supernatants of AVE was significantly up-regulated. Calcium deposits have been observed only after pro-fibrotic/pro-calcific cultivation. Although, western blot analysis has proven that the used OPN is phosphorylated on tyrosine, serine and threonine phosphorylation sites, OPN did neither inhibit the pronounced effects on morphological changes, gene and protein expression of fibrosis markers nor calcium deposition and ALP activity.

Conclusion: Our data indicate that OPN does not mediate anti-fibrotic and anti-calcific effects in ovine AVE. Thus, future studies should assess whether the promising effects of phosphorylated OPN shown on smooth muscle cells in classical two-dimensional cell culture models can be transferred to three-dimensional cell culture models of VIC.