Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice

Oxidative Medicine and Cellular Longevity, Aug 2016

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as . Cultured cells condition their media to reproduce physiological , but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the media before and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellular than young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

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Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice

Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice Walter H. Watson,1,2 Tom J. Burke,1 Igor N. Zelko,3,4 Edilson Torres-González,4,5 Jeffrey D. Ritzenthaler,4 and Jesse Roman2,4,5 1Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Louisville School of Medicine, Louisville, KY, USA 2Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA 3Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Louisville, KY, USA 4Department of Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, University of Louisville School of Medicine, Louisville, KY, USA 5Robley Rex Veterans Affairs Medical Center, Louisville, KY, USA Received 5 May 2016; Accepted 7 August 2016 Academic Editor: Jean-Claude Lavoie Copyright © 2016 Walter H. Watson et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as . Cultured cells condition their media to reproduce physiological , but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the media before and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellular than young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment. 1. Introduction Aging is associated with increased incidence of respiratory disorders, and elderly patients represent a disproportionate number of afflicted individuals with pneumonia, acute lung injury, and lung fibrosis, among other lung disorders [1–5]. This association has also been noted in experimental models of lung disease. For example, senescent rodent lungs are more susceptible to lung injury in the setting of mechanical ventilation, ozone exposure, and pulmonary infection [6–10]. Furthermore, intratracheal instillation of lipopolysaccharide results in the exaggerated expression of proinflammatory cytokines in senescent animals when compared to young controls [9, 11]. Senescent lungs are also more susceptible to bleomycin-induced lung injury [12, 13]. Together, these studies point to the enhanced susceptibility of the senescent lung to injury, but little is known about the factors responsible for this susceptibility. Several mechanisms have been proposed to explain the abovementioned observations including increased oxidative stress and free radical damage, a decline in immune responses, and alterations in stem cell/progenitor cell differentiation potential [14–16]. Mitochondrial dysfunction has also been implicated in aging since the coordination between nuclear and mitochondrial communication during aging appears to be affected [17]. There is also much literature showing alterations in lung structure and function in the aging lung [18–21]. Consistent with this, we previously reported that aging murine lungs harvested from old mice are characterized by increased expression of fibronectin and collagen matrix mRNAs and by induction of the profibrotic factor, transforming growth factor β [22]. Thus, active matrix remodeling may account for the subtle changes observed in lung structure as well as the decline in lung function observed in the elderly [23] and might render the host susceptible to disrepair after lung injury [22]. Another abnormality associated with aging is a shift in the redox states of plasma thiol-disulfide redox couples [24, 25]. Cysteine (Cys) and its disulfide cystine (CySS) constitute the major small molecular weight thiol-disulfide redox couple in extracellular compartments. The redox state of the Cys/CySS couple, expressed as calculated from the Nernst equation, is about −80 mV in the plasma of healthy young adults [26] but is more oxidized in older individuals [24]. The mechanisms that regulate a (...truncated)


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Walter H. Watson, Tom J. Burke, Igor N. Zelko, Edilson Torres-González, Jeffrey D. Ritzenthaler, Jesse Roman. Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice, Oxidative Medicine and Cellular Longevity, 2016, 2016, DOI: 10.1155/2016/1561305