Citations

Rewarming shock (acute cardiac failure) during resuscitation from profound hypothermia is partially ameliorated by low dose epinephrine (EPI). However, hypothermia‐induced cardiac Ca 2+ overload persists with rewarming. The reactive oxygen species (ROS) scavenger superoxide dismutase (SOD) protects isolated hearts from hypothermia‐induced Ca 2+ overload. In electrically stimulated rat cardiac myocytes loaded with Ca 2+ indicators fluo‐3 (cytosolic) and rhod‐2 (mitochondria) and imaged using confocal microscopy, we investigated the effects of EPI and SOD on cytosolic vs. mitochondrial Ca 2+ during rewarming. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h to 37 °C. EPI 0.125 μg/ml was administered from 27 °C upwards, and SOD 0.1 mg/ml from 15 °C upwards during rewarming. Control cells did not receive drugs. Other cells received EPI alone. Hypothermia resulted in elevated cytosolic and mitochondrial Ca 2+ . Compared to normothermia, mitochondrial Ca 2+ uptake and release was slowed by hypothermia, and not immediately reversed with rewarming. EPI prolonged mitochondrial Ca 2+ overload during rewarming. With SOD, mitochondrial as well as cytosolic Ca 2+ were comparable to controls during hypothermia and rewarming. These data suggest that during rewarming, mitochondrial Ca 2+ buffering remains impaired, and that ROS may play a role in Ca 2+ overload. Administration of EPI may exacerbate mitochondrial dysfunction, potentially worsening rewarming shock. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

The mechanisms underlying rewarming shock (cardiac failure during rewarming from profound hypothermia) are not known. Increased myocardial Ca 2+ levels and altered mitochondrial ultrastructure have been reported. We hypothesized that mitochondrial buffering of cytosolic Ca 2+ contributes to cardiac dysfunction. Enzymatically‐dissociated rat cardiac myocytes loaded with Ca 2+ indicators fluo‐3 (cytosolic) and rhod‐2 (mitochondrial) were electrically stimulated (0.5 Hz) and visualized using real time confocal microscopy. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h back to 37 °C. Hypothermia increased baseline mitochondrial Ca 2+ (134% of normothermic control), which returned to control levels after rewarming. In contrast, rewarming elevated cytosolic Ca 2+ significantly compared to controls (126% vs. 94%). During hypothermia both cytosolic and mitochondrial peak Ca 2+ , time to peak, and time to 50% decay increased by 135%, 150% and 200%, respectively, but returned to control levels during rewarming. These data indicate that during hypothermia, mitochondria buffer cytosolic Ca 2+ , and during rewarming, mitochondrial Ca 2+ is redistributed to the cytosol, leading to continued elevation in cytosolic Ca 2+ . The temporary Ca 2+ overload in mitochondria may further affect subsequent energy production and myocyte function. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

Pharmacologic stimulation of a G protein–coupled receptor preferentially expressed on fibroblasts reverses experimental lung and liver fibrosis.

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)‐β, which upon activation down‐regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER‐α vs. ER‐β activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E 2 , an ER‐α agonist [propylpyrazoletriol (PPT)], and an ER‐β agonist [WAY‐200070 (WAY)] with TNF‐α or platelet‐derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT‐PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP‐1) and NF‐κB. TNF‐α or PDGF significantly ( P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF‐α‐ or PDGF‐induced ECM gene expression in ASM cells was significantly reduced with WAY ( P < 0.001). Moreover, WAY significantly down‐regulated the activation of NF‐κB( P < 0.001) and AP‐1 ( P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER‐β diminishes ECM deposition via suppressing the NF‐κB pathway activity and might serve as a novel target to blunt airway remodeling.—Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen‐receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF‐κB pathway. FASEB J. 33, 13935‐13950 (2019). www.fasebj.org

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERβ, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]iregulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]iresponse to histamine, whereas ERβ activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERβ activation was more effective than 17β-estradiol in reducing [Ca2+]iresponses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERβ were mediated partially via L-type calcium channel inhibition and increased Ca2+sequestration by sarcoplasmic reticulum.Overall, these data highlight the differential signaling of ERα and ERβ in ASM during inflammation. Specific ERβ activation reduces [Ca2+]iin the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.

Hyperoxia exposure in premature infants increases the risk of subsequent lung diseases, such as asthma and bronchopulmonary dysplasia. Fibroblasts help maintain bronchial and alveolar integrity. Thus, understanding mechanisms by which hyperoxia influences fibroblasts is critical. Cellular senescence is increasingly recognized as important to the pathophysiology of multiple diseases. We hypothesized that clinically relevant moderate hyperoxia (<50% O2) induces senescence in developing fibroblasts. Using primary human fetal lung fibroblasts, we investigated effects of 40% O2 on senescence, endoplasmic reticulum (ER) stress, and autophagy pathways. Fibroblasts were exposed to 21% or 40% O2 for 7 days with etoposide as a positive control to induce senescence, evaluated by morphological changes, β-galactosidase activity, and DNA damage markers. Senescence-associated secretory phenotype (SASP) profile of inflammatory and profibrotic markers was further assessed. Hyperoxia decreased proliferation but increased cell size. SA-β-gal activity and DNA damage response, cell cycle arrest in G2/M phase, and marked upregulation of phosphorylated p53 and p21 were noted. Reduced autophagy was noted with hyperoxia. mRNA expression of proinflammatory and profibrotic factors (TNF-α, IL-1, IL-8, MMP3) was elevated by hyperoxia or etoposide. Hyperoxia increased several SASP factors (PAI-1, IL1-α, IL1-β, IL-6, LAP, TNF-α). The secretome of senescent fibroblasts promoted extracellular matrix formation by naïve fibroblasts. Overall, we demonstrate that moderate hyperoxia enhances senescence in primary human fetal lung fibroblasts with reduced autophagy but not enhanced ER stress. The resulting SASP is profibrotic and may contribute to abnormal repair in the lung following hyperoxia.