Abstract 4368252: A Novel Role for ANPEP in Osteogenic Signaling and Aortic Valve Calcification

Introduction: Calcific aortic valve disease (CAVD) is one of the most common valvular diseases, predominantly characterized by progressive calcification of the aortic valve. Although minimally invasive treatments such as transcatheter aortic valve replacement (TAVR) have advanced rapidly, there remains a lack of effective strategies to delay or reverse the progression of CAVD. This study investigates underlying molecular mechanisms of CAVD to explore new therapeutic avenues. Methods: Three publicly available human bulk RNA-seq datasets related to CAVD were analyzed. Genes upregulated across all three datasets were identified and intersected to find shared targets. Aortic valve tissues from healthy individuals and CAVD patients were collected, and Western blotting was used to validate expression levels of candidate genes. In vitro, small interfering RNA (siRNA) was used to knock down the target gene in valvular interstitial cells (VICs), evaluating its effect on calcification markers. In vivo, we generated ANPEP knockout mice (ANPEP-/-/ApoE-/-) via breeding and subjected them to a high-fat diet to induce valve calcification. Alizarin Red S staining was performed to assess the extent of calcification. Results: Only two genes—ANPEP and CTHRC1—were commonly upregulated across all datasets. CTHRC1 has been previously studied in CAVD, while ANPEP, a zinc-dependent type II metalloprotease primarily expressed on the cell membrane, has not. Western blot analysis confirmed significantly increased ANPEP expression in calcified human valve tissue. siRNA-mediated knockdown of ANPEP in VICs led to marked reductions in calcification markers such as BMP2 and RUNX2, suggesting its role in calcification. In vivo, ANPEP-deficient mice showed significantly less aortic valve calcification, aligning with in vitro findings. Conclusion: These results indicate that ANPEP plays a critical role in the progression of CAVD and may serve as a promising target for the diagnosis and treatment of aortic stenosis due to valve calcification.