Involvement of proprotein convertase subtilisin/kexin type 9 in aortic valve calcification

Background and Aims: Calcific aortic valve stenosis (CAVS) affects 3% of the general population lacking pharmacological treatment. Inhibiting proprotein-convertase-subtilisin/kexin-type9 (PCSK9) has demonstrated promise in impeding calcium deposition on human aortic valve interstitial cells (VIC), supporting the potential benefit of PCSK9 therapies to mitigate CAVS progression. Our investigation seeks to elucidate PCSK9 involvement in the aortic leaflets calcification processes. Methods: Human leaflets were collected and VICs isolated to perform histological analysis, enzyme-linked immunosorbent assay (ELISA), and calcification assays. CRISPR/Cas9 technology was used to generate PCSK9 overexpressing immortalized human VICs (PCSK9+iVIC). PCSK9 knockout (KO) and wild-type (WT) C57BL/6J mice were followed for 12/18 months under standard or high-fat diet and underwent Doppler-echocardiography and micro-computed tomography (μCT). Results: During CAVS progression, a PCSK9 increment was evident both via immunohistochemistry (log2FC: sclerotic +1.8±0.7 and stenotic +2.7±0.5; ANOVA p=0.0008) and ELISA (FC: sclerotic +9.8±1.6 and stenotic +11.13±2.3; ANOVA p=0.0012). Both in normal and osteogenic conditions, sclerotic and stenotic human VICs calcify more than controls (p<0.001). The correlation between calcium and secreted PCSK9 increased parallel to disease progression (control rs=0.04, p=0.8; sclerotic rs=0.5, p=0.004; stenotic rs=0.6, p=0.0003). PCSK9+iVICs showed an increased calcification content starting at day 7 (p<0.05) and increasing up to day 21 (p<0.0001) in pro-osteogenic stimulus compared to iVICs. In the in vivo model, before sacrifice, we observed a lower peak aortic jet velocity in KO mice compared with WT mice (-10%; p<0.05), suggesting a propensity of PCSK9 to promote aortic valve remodelling. Histological analysis showed aortic valve remodelling in WT mice compared with KO ones. However, μCT calcification content did not differ between groups. Conclusions: Our study highlights that PCSK9 is upregulated in CAVS and correlates with calcium deposition in human VICs. Moreover, PCSK9 overexpression enhances calcification in vitro, while PCSK9 deficiency attenuates aortic valve remodeling in vivo.