Abstract 13063: Clonal Hematopoiesis Associated With TET2 and DNMT3A Gene Driver Mutations Accelerates Aortic Valve Calcification in vitro and in vivo

Abstract

Calcific aortic valve disease (CAVD) is the most common age-related heart valve disease, with no medical therapy to halt progression. Age is associated with the enrichment of somatic mutations in hematopoietic stem cells leading to clonal hematopoiesis (CH). CH mutations promote inflammation, occur in more than 30% of patients with severe CAVD and CAVD patients harboring CH mutations have a worse prognosis. Single-cell RNA-sequencing of immune cells (n=127120 cells) of CAVD patients with CH-mutations showed strong proinflammatory and M1-like macrophage gene signatures (CD38, CXCL10) along with genes associated with calcification (S100A9, RUNX2, Oncostatin M=OSM) suggesting that CH might be causally involved in CAVD. Indeed, silencing of TET2 or DNMT3A in macrophages in vitro induce the prototypic osteoblastic transcription factor RUNX2 and paracrine acting genes such as OSM and S100A9, which can promote vascular calcification. Secreted factors from TET2 or DNMT3A-silenced macrophages induced osteoblastic differentiation as demonstrated by elevated calcium deposition and genes involved in the RUNX2 signaling pathway (COL1A2, ALP), which could be ablated by silencing of OSM in CH-macrophages. Atheroprone Ldlr -/- mice receiving TET2 -/- bone marrow transplants mice showed increased total calcified area along (1.49-fold) with increased numbers of calcification deposits (2.33-fold) as evidenced by von Kossa staining. Increased myeloid derived OSM and S100A9 was found in the valves of TET2 -/- BMT mice with S100A9 strongly co-occurring with OSM. This study provides insights into the development of incident CAVD for patients with CH mutations and therefore yields plausible interventions for nearly one third of CAVD cases to slow or stop disease progression.