AMBP protects against aortic valve calcification by inhibiting ERK1/2 and JNK pathways mediated by FHL3

Rationale: Calcific aortic valve disease (CAVD) is a progressive disorder characterized by aortic valve (AV) calcification and fibrosis. Despite advances in our understanding of CAVD pathogenesis, no drug has proven effective in preventing AV calcification. The aim of this study was to identify the key pathogenic genes in CAVD and elucidate mechanisms that may guide development of new targeted therapies. Methods: A CAVD model was established in ApoE^-/- mice by administering a high-cholesterol diet for 24 weeks. An adeno-associated virus was used to induce alpha-1-microglobulin/bikunin precursor (AMBP) overexpression. RNA sequencing, quantitative real-time polymerase chain reaction, western blotting, immunofluorescence, histopathology, and echocardiography were performed to assess AV function. The mechanism of interaction between AMBP and four-and-a-half LIM domain protein 3 (FHL3) was explored using bioinformatics analyses, co-immunoprecipitation, and AlphaFold3-based simulations of crystal structures. Results: RNA sequencing identified AMBP as a key regulator of CAVD. AMBP was increased in calcified AV from CAVD patients and high cholesterol diet (HCD)-induced ApoE^-/- mice. In vivo, AMBP overexpression significantly reduced HCD-induced AV calcification and fibrosis. In vitro, AMBP knockdown elevated osteogenic markers, RUNX2 and OSTERIX, and promoted calcium deposition in valvular interstitial cells induced by osteogenic medium (OM), whereas AMBP overexpression reversed these effects. Mechanistically, AMBP inhibited OM-induced phosphorylation of ERK1/2 (P-ERK1/2) and JNK (P-JNK) by competitively binding to the zinc finger domain of FHL3. This interaction disrupted the protective role of FHL3 in preventing ubiquitin-proteasome-mediated degradation of P-ERK1/2 and P-JNK. P-ERK1/2 and P-JNK inhibitors and agonists confirmed that the protective effects of AMBP against CAVD were mediated via these pathways in vivo and in vitro. Conclusions: AMBP protects valvular interstitial cells from osteoblastic differentiation and calcium deposit accumulation, thereby alleviating AV calcification. This study sheds additional light on the pathogenesis of CAVD and potential new therapeutic approaches.