Aortic valve calcification is promoted by clonal hematopoiesis associated with DNMT3A and TET2 gene driver mutations

Abstract Background Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations is associated with coronary heart disease and worse prognosis among patients with aortic valve stenosis (AVS) and inflammation. However, it is unknown what role CH plays in the pathogenesis of AVS. AVS is the most common age-related heart valve disease, with no medical therapy to halt progression. Methods and Results Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), with mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, many associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) and paracrine pro-calcific factors (oncostatin M (OSM), S100A9), suggesting that CH might be causally involved in AVS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced secretomes induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic conversion could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving TET2-/- bone marrow transplants demonstrated increased total calcified area (1.49-fold) along with increased numbers of calcification deposits (2.33-fold) as evidenced by von Kossa staining, along with enhanced OSM and S100A9 levels. Conclusion This is the first study to demonstrate that somatic CH-driver mutations in monocytes may accelerate AVS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the progression of AVS in patients harboring CH mutations.