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Cyb5R3 an External Mitochondrial NADH - Dependent Reductase Confers Protection Against Lung Fibrosis
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A4351 - Cyb5R3 an External Mitochondrial NADH - Dependent Reductase Confers Protection Against Lung Fibrosis
Author Block: M. Bueno, B. G. Mays, K. Fiedler, A. Nasser, S. J. Shulkowski, A. Potkonjak, J. Pogue, J. T. Carter, S. Hahn, A. C. Straub, A. L. Mora; University of Pittsburgh, Pittsburgh, PA, United States.
Rationale: Nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase 3 (Cyb5R3) or methemoglobin reductase is a flavoprotein known for its ability to transfer electrons from its NADH domain through the small heme protein partner Cyb5 (cytochrome b5) to a final electron acceptor. Mitochondrial membrane-bound Cyb5R3 regulates several biological reduction reactions and, in doing so, regulates the ratio of pyridine nucleotides (NAD+/NADH), a key regulator of age-related biochemical changes. Levels of NAD+ decrease with age. However, the role of the pyridine nucleotide ratio in the pathobiology of Idiopathic Pulmonary Fibrosis (IPF), an age-associated disease, is still unknown. Methods: Lungs from young, old and IPF donors were lysate to test Cyb5R3 levels and concentrations of NAD+ and NADH. Cyb5R3 flox mice (Cyb5R3 fl/fl) and mice carrying a doxycycline-inducible Cre-recombinase under the control of a regulatory sequence of the SP-C gene were bred. To promote recombination and generate a homozygous conditional type II lung epithelial cells CYB5R3 knockout mice (Cyb5R3 spc-KO), mice were fed doxycycline chow (625 mg/kg, Envigo) for 2 weeks before injury induction. Young 3 month old Cyb5R3 fl/fl and Cyb5R3 spc-KO mice were infected intranasally with gammaherpesvirus 68 (MHV68) to evaluate inflammatory and fibrotic responses in the lung. Results: Aged lungs and IPF lungs show lower levels of Cyb5R3 and lower levels of NAD/NADH. Conditional deletion of Cyb5R3 type II in lung epithelial cells resulted in increased susceptibility to virus-induced lung fibrosis. Cyb5R3 spc-KO mice showed collagen deposition by Masson trichrome by 15 days post infection (dpi) and corroborated by hydroxyproline assay. Additionally, collagen, fibronectin and TGFβ expression was significantly higher in the Cyb5R3 spc-KO mice at15dpi when compared to the Cyb5R3 fl/fl control mice. Inflammatory responses were also more severe in the Cyb5R3 spc-KO mice, as determined by BAL cell counts and the changes in mRNA levels of the cytokines TNFα, and IL6. Conclusions: Altogether, these data suggest a possible role for Cyb5R3 in the fragility of AECIIs and the age related susceptibility to lung fibrosis. They also suggest that strategies designed to restore the ratio of pyridine nucleotides(NAD+/NADH) or modulated Cyb5R3 expression might lessen severity of pulmonary fibrosis, especially in the context of the aging lung. Funding: Vascular Medicine Institute
Author Block: M. Bueno, B. G. Mays, K. Fiedler, A. Nasser, S. J. Shulkowski, A. Potkonjak, J. Pogue, J. T. Carter, S. Hahn, A. C. Straub, A. L. Mora; University of Pittsburgh, Pittsburgh, PA, United States.
Rationale: Nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase 3 (Cyb5R3) or methemoglobin reductase is a flavoprotein known for its ability to transfer electrons from its NADH domain through the small heme protein partner Cyb5 (cytochrome b5) to a final electron acceptor. Mitochondrial membrane-bound Cyb5R3 regulates several biological reduction reactions and, in doing so, regulates the ratio of pyridine nucleotides (NAD+/NADH), a key regulator of age-related biochemical changes. Levels of NAD+ decrease with age. However, the role of the pyridine nucleotide ratio in the pathobiology of Idiopathic Pulmonary Fibrosis (IPF), an age-associated disease, is still unknown. Methods: Lungs from young, old and IPF donors were lysate to test Cyb5R3 levels and concentrations of NAD+ and NADH. Cyb5R3 flox mice (Cyb5R3 fl/fl) and mice carrying a doxycycline-inducible Cre-recombinase under the control of a regulatory sequence of the SP-C gene were bred. To promote recombination and generate a homozygous conditional type II lung epithelial cells CYB5R3 knockout mice (Cyb5R3 spc-KO), mice were fed doxycycline chow (625 mg/kg, Envigo) for 2 weeks before injury induction. Young 3 month old Cyb5R3 fl/fl and Cyb5R3 spc-KO mice were infected intranasally with gammaherpesvirus 68 (MHV68) to evaluate inflammatory and fibrotic responses in the lung. Results: Aged lungs and IPF lungs show lower levels of Cyb5R3 and lower levels of NAD/NADH. Conditional deletion of Cyb5R3 type II in lung epithelial cells resulted in increased susceptibility to virus-induced lung fibrosis. Cyb5R3 spc-KO mice showed collagen deposition by Masson trichrome by 15 days post infection (dpi) and corroborated by hydroxyproline assay. Additionally, collagen, fibronectin and TGFβ expression was significantly higher in the Cyb5R3 spc-KO mice at15dpi when compared to the Cyb5R3 fl/fl control mice. Inflammatory responses were also more severe in the Cyb5R3 spc-KO mice, as determined by BAL cell counts and the changes in mRNA levels of the cytokines TNFα, and IL6. Conclusions: Altogether, these data suggest a possible role for Cyb5R3 in the fragility of AECIIs and the age related susceptibility to lung fibrosis. They also suggest that strategies designed to restore the ratio of pyridine nucleotides(NAD+/NADH) or modulated Cyb5R3 expression might lessen severity of pulmonary fibrosis, especially in the context of the aging lung. Funding: Vascular Medicine Institute
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