Sirtuins in Lung Aging and Fibrosis

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Human fibrotic disorders affect many organ systems including heart, blood vessels, kidney, liver and lung, accounting for excessive disease burden in the U.S., including among our Veterans Administration (VA) patient population. The most common fibrotic lung disease, idiopathic pulmonary fibrosis (IPF), is characterized by excessive scar tissue formation and irreversible destruction of the lung parenchyma, resulting in gas-exchange abnormalities and ultimately respiratory failure. Aging is strongest risk factor for IPF; however, the cellular/molecular mechanisms that account for the age-associated predilection to fibrotic disease are only beginning to be explored. Improved understanding of the role of aging in this disease will aid in the development of novel therapies with greater clinical efficacy. In support of this VA Merit Award renewal application, we have made several recent contributions to our understanding of aging mechanisms in lung injury and repair mechanisms. Human subjects with IPF express low levels of the mitochondrial sirtuin, SIRT3, in myofibroblastic foci and in ex-vivo fibroblasts isolated from IPF lungs. This down-regulation of SIRT3 is replicated in an aging model of non-resolving fibrosis in mice; while fibrosis in young mice largely resolves over 2-4 months post-bleomycin, aged mice manifest persistent fibrosis. Our data indicate that this capacity for fibrosis resolution in young mice is associated with recovery of SIRT3 levels in the late reparative phase of lung injury, while this is absent in aged mice. However, re-constitution of SIRT3 (via lung-targeted non-viral cDNA plasmid delivery) restores the capacity for fibrosis resolution in aged mice. This protective effect is associated with in-vivo activation of FoxO3a, evidenced by higher levels of nuclear FoxO3a. Both cellular senescence and the pro-fibrotic cytokine, transforming growth factor-β1 (TGF- β1) induce a down-regulation of SIRT3 and FoxO3a, events that lead to mitochondrial dysfunction, senescence and apoptosis resistance of lung fibroblasts. Our studies also support the possibility that modulation of SIRT3 in alveolar macrophages regulates fibroblast activation of FoxO3a by a paracrine mechanism. Additionally, the histone acetyltransferases, p300 and cyclic AMP-response element binding protein (CREB)-binding protein (CBP), are implicated in the epigenetic down-regulation of SIRT3 by TGF-β1 and cellular senescence. Impaired activation of the SIRT3-FoxO3a signaling axis promotes a senescent and apoptosis-resistant fibroblast phenotype, which drives persistent/non-resolving fibrosis. The central hypothesis to be tested in this grant proposal is that persistent/non-resolving fibrosis associated with aging is mediated by impaired activation of Foxo3a in fibroblasts, either by SIRT3 deficiency in fibroblasts themselves or by macrophage-derived paracrine mechanisms, contributes to mitochondrial dysfunction, senescence and apoptosis resistance of lung fibroblasts. Our specific aims are to: (1) elucidate p300/CBP-dependent epigenetic regulation of SIRT3 in lung fibroblasts and alveolar macrophages that mediate pro-resolution phenotypes; (2) investigate the role of the SIRT3-FoxO3a signaling axis in restoring mitochondrial bioenergetics and apoptosis susceptibility to senescent/fibrotic lung fibroblasts; and (3) determine whether reconstitution of SIRT3 by genetic/pharmacologic approaches promotes fibrosis resolution in aged mice via the activation of FoxO3a in lung fibroblasts. The completion of the Aims in this proposal will: (a) uncover epigenetic mechanisms that control SIRT3 expression with cellular senescence/aging; (b) provide mechanistic insights into the role of SIRT3-FoxO3a signaling in the regulation of mitochondrial bioenergetics and cellular plasticity/fate; and (c) elucidate macrophage-fibroblast crosstalk and SIRT3 regulation of pro-resolving macrophages; and (d) provide proof-of-concept that activation of the SIRT3-FoxO3a signaling pathway promotes fibrosis resolution, leading to new therapeutic approaches for progressive lung diseases.