Dysregulation of ATPases Promotes the Degeneration of Aortic Valves

 

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Objectives: Calcific aortic valve disease (CAVD) has been identified as a slow and progressive, but also as an active and regulated process involving creation of calcium nodules, lipoprotein accumulation, and chronic inflammation. ATPases are a class of enzymes that catalyze the decomposition of ATP into ADP and have been considered as attractive drug targets. The key enzymes of the purinergic signaling system profoundly affect the degeneration of aortic valves (AV), but the role of related ATPases is still unknown. Using valvular interstitial cells (VICs) and AV cusps tissue, we aimed to clarify how dysregulation of ATPases influences the degeneration process of AVs.

Methods: Ovine VICs, as well as defined biopsies of AV cusps, were treated in vitro under pro-degenerative conditions (β-glycerophosphate and CaCl2) with inhibitors of ATPases (suramin and vanadate). Calcium deposition was evaluated by a commercial assay and alizarin red staining. Expression of mRNA was analyzed by semiquantitative RT-PCR. Expression of alpha smooth muscle actin (α-SMA) and vimentin (VIM) was analyzed by Western blot and immunofluorescence microscopy. Morphological changes of the extracellular matrix (ECM) of AV cusps were assessed histologically by hematoxylin/eosin and Movat pentachrome.

Results: Inhibition of ATPases resulted in distinct pro-degenerative ramifications of VIC cultures. Gene and protein expression of α-SMA was increased after treatment with suramin (p < 0.05) and decreased after treatment with vanadate (p < 0.05). Furthermore, suramin inhibited protein expression of VIM under pro-degenerative conditions. Moreover, treatment with both ATPase inhibitors significantly altered mRNA expression of enzymes of the purinergic signaling system. In biopsies of native AVs, inhibition of ATPases resulted in elevated calcium and phosphate accumulation. Increased ECM remodeling as well as increasing structural disorganization of AV cusps tissue confirmed a strong pro-degenerative effect. Both treatments increased the expression of α-SMA (p < 0.05), while VIM was decreased after treatment with suramin (p < 0.05).

Conclusion: Inhibition of ATPases resulted in pro-degenerative effects in VICs and AV cusps tissue. Our findings suggest that transmembrane ATPases play a profound role in the process of initiation and propagation of degenerative events in CAVD. Regulation of ATP-conversing enzymes could be a promising approach to prevent CAVD progression.