Abstract Rationale Tissue regenerative capacity declines with aging and is associated with a propensity for fibrosis. Prior studies have shown that the tricarboxylic acid (TCA) cycle metabolite, oxaloacetate (OAA), induces pro-regenerative responses in association with activation of the Wnt/β-catenin pathway in a murine model of digit regeneration. In this study, we aimed to determine: (a) whether OAA modulates injury repair responses in the lung; and (b) mechanisms by which the Wnt/β-catenin pathway is activated by OAA. Methods Young (2 months) and aged (18 months) C57BL/6 mice were administered bleomycin. On day 14, mice received daily intraperitoneal 0.5 g/kg OAA injections until day 35 when lungs were collected for histology. Body weight measurement and micro-computerized tomography (µ-CT) were assessed at weekly intervals. Human lung fibroblasts (IMR90 cells) were passaged to a low population doubling level (LPDL) and high population doubling level (HPDL) as a model of replicative senescence. LPDL and HPDL cells were treated with 2 mM OAA, western blot and mRNA expression analysis focusing on the Wnt/β-catenin pathway were performed. Furthermore, we silenced the acetyltransferase EP300, which may mediate acetylation/activation of β-catenin, and assessed Wnt pathway activation. Results OAA treatment in young mice challenged with bleomycin demonstrated no significant changes in fibrosis resolution while aged mice administered OAA developed worse fibrosis based on µ-CT assessments and collagen deposition quantitated by histology. DEG analysis of HPDL-LPDL lung fibroblasts revealed differential expression of Wnt ligands. Both LPDL and HPDL lung fibroblasts treated with OAA (2 mM) induced β-catenin acetylation (lys49), in association with phosphorylation of AKT and GSK3β; silencing of EP300 abrogated these OAA induced signaling events. Conclusion Our studies demonstrate that OAA worsens fibrosis in aged mice subjected to bleomycin-induced lung injury in-vivo and activates the Wnt/β-catenin pathway in lung fibroblasts ex-vivo. Aberrant activation of this pathway in aging may be mediated by differential expressions of Wnt ligands or ligand-independent metabolic derangements that activate β-catenin. This model may serve to elucidate how a pro-regenerative pathway may be co-opted to pro-fibrotic responses in the context of an aging metabolic landscape. This abstract is funded by: Supported by NIH grants, R01 HL173154, R01 HL139617, R01 HL151702, and the U.S. Department of Veterans Affairs Merit Award I01BX003056.
Jaramillo et al. (Fri,) studied this question.