|Topic:||27. Structure, Function and Mechanism / Adult / Aging / Respiratory Cell and Molecular Biology (RCMB)|
|Authors:||W.H. Watson, J.D. Ritzenthaler, T.J. Burke, I.N. Zelko, E. Torres-Gonzalez, J. Roman; Louisville, KY/US|
Rationale: The aging lung is characterized by increased susceptibility to injury, but the mechanisms involved remain uncertain. We have found that aging murine lungs show alterations in the relative composition of the extracellular matrix (ECM) without overt changes in architecture, a process termed transitional remodeling that might render hosts susceptible to injury/disrepair. Aging is also associated with oxidation of the plasma cysteine (Cys)/cysteine (CySS) redox state (expressed as Eh Cys/CySS), and we hypothesized that lung fibroblasts influence this process, which might be linked to transitional remodeling.
Methods: Primary lung fibroblasts harvested from young (2 months old) and old (24 months old) C57BL/6 mice were evaluated for their expression of ECM and the phenotypic marker Thy-1 via RT-PCR. The ability of these cells to maintain their redox state was examined by culturing young and old lung fibroblasts in media with different Eh Cys/CySS redox states, and Eh Cys/CySS was measured in conditioned media as a function of time in culture. Expression of genes related to redox regulation and fibroblast function was measured by microarray and RT-PCR. Cystine transport across the cell membrane was induced and inhibited by sulforaphane and sulfasalazine, respectively.
Results: Aging lung fibroblasts showed increased mRNA expression for fibronectin EDA, type I collagen, and plasminogen activator inhibitor-1, and a decrease in Thy-1 when compared to young cells, consistent with our prior work (Sueblinvong et al., Am J Med Sci, 2012). Old fibroblasts also produced a more oxidizing extracellular Eh Cys/CySS than young fibroblasts reminiscent of the oxidized Eh Cys/CySS noted in elderly individuals. This oxidation was associated with a slower rate of reduction of CySS to Cys. Of the 36 antioxidant and thiol-disulfide regulating enzymes analyzed, only the Slc7a11 subunit of the xCT cystine-glutamate transporter and extracellular superoxide dismutase (Sod3) were down-regulated in lung fibroblasts from old mice. In contrast, there were many changes in ECM components between old and young. Induction of expression of Slc7a11 by sulforaphane was associated with a more reducing Eh Cys/CySS, and inhibition of Slc7a11 activity by sulfasalazine led to a more oxidizing Eh Cys/CySS.
Conclusion: Lung fibroblasts play an active role in controlling their extracellular redox environment, and aging re-programs them to show a pro-fibrotic phenotype and maintain a more oxidizing set point than young fibroblasts, perhaps through alterations in the expression of Slc7a11.