Abstract P3070: Aortic Valve Calcification Is Accelerated By DNMT3A And TET2 Gene Driver Mutations Associated With Clonal Hematopoiesis

Abstract

Background: Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations associates with heightened inflammation and a worse prognosis among patients with severe calcific aortic valve stenosis (AS). However, it is unknown what role CH plays in the pathogenesis of AS. AS 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), having a 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, which were associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) along with paracrine pro-calcific factors (Oncostatin M (OSM), S100A9), signifying that CH might be causally involved in AS. 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 supernatants 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 transformation could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving Tet2-/- bone marrow transplants demonstrated increased total calcified area (p<0.05, 1.49-fold) along with increased numbers of calcification deposits (p<0.05, 2.33-fold) as shown by von Kossa staining, along with enhanced OSM and S100A9 levels. Conclusion: This is the first study to show that somatic CH-driver mutations in monocytes may accelerate AS. 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 development of AS in patients harboring CH mutations.