Abstract Body:
Introduction:
Recent studies suggest that SGLT2 inhibitors may improve the prognosis of aortic stenosis (AS) by limiting calcification. Thickening and calcification of valve leaflets during AS is caused by osteoblastic transformation of valvular interstitial cells (VICs).
Aim:
This study aims to investigate whether empagliflozin (Empa) has a direct effect on osteoblastic transformation process of human VICs in vitro.
Methods:
hVICs were cultured in osteogenic medium (OM) and treated with or without Empa (30 nM or 150 nM) for 5 or 21 days. In a separate experiment, cells were cultured in OM under normal or high glucose (Glu) (25 mM) with or without Empa. Calcification was assessed by Alizarin Red staining. Expression of osteogenic markers (BMP2, RUNX2) and kinase signaling pathways was evaluated by Western blotting. Osteocalcin (OC), osteopontin (OP), and inflammatory mediators (IL-6, IL-1β) were quantified by ELISA.
Results:
Calcification was almost 6-fold higher in both OM and OM+Glu after 21 days compared with the control group (Alizarin concentration [Conc]: 0.13 vs 0.64 vs 0.65 mM, p<0.001). OC and OP levels increased significantly in both the OM and OM+glucose groups, accompanied by RUNX2 upregulation. At day 5, BMP2 expression increased 3.5-fold in both groups, along with a 5-fold increase in phospho/total ERK1/2. In contrast, phospho/total Akt and phospho/total p38 showed minor changes, and inflammatory mediators remained unchanged. Empa (30 or 150 nM) did not reduce calcification in VICs cultured in OM (Conc:1.30 and 0.95 vs 0.64 mM) or OM+glucose (Conc:0.79 and 0.80 vs 0.65 mM). Under normoglycaemic OM conditions, Empa even increased calcification after 21 days, and was associated with early activation of ERK1/2 and Akt without affecting BMP2 expression. In OM+glucose conditions, Empa did not affect either BMP2 expression or ERK1/2 activation.
Conclusion:
OM induced osteoblastic transformation of VICs via BMP2/ERK pathway, whereas high glucose had no direct effect. Empa did not inhibit calcification in isolated hVICs and, under normoglycaemic conditions, even worsened it, with early ERK and Akt activation. Further studies are needed to explore the effects of Empa on other elements of the calcification process.