Thompson, Michael A

ABSTRACT Supplemental oxygen (hyperoxia), often provided to premature infants, can disrupt lung growth and contribute to development of neonatal and pediatric lung diseases, necessitating understanding of underlying mechanisms. We previously showed that even moderate hyperoxia (< 60% O 2 ) induces detrimental cellular senescence in 18–22 weeks human fetal airway smooth muscle (fASM), a key cell type in airway contractility and remodeling. In this study, we examined the ability of senotherapeutics Fucoidan and Dasatinib (D) + Quercetin (Q) (D + Q) to mitigate adverse effects of hyperoxia and assessed the preventive effect of mitochondrial antioxidants (MitoQ). fASM cells exposed to normoxia (21% O 2 ) or hyperoxia (50% O 2 ) were treated with Fucoidan [100 μg/mL], D + Q [250 nM + 375 nM] or MitoQ [100 nM] and assessed for senescence markers, senescence‐associated secretory profile (SASP), extracellular matrix (ECM) deposition, and cell viability. Our results showed that moderate hyperoxia increases senescence markers and SASP. Fucoidan decreased p21 expression and inhibited SASP release without inducing cell death, while D + Q decreased β‐galactosidase (β‐Gal) activity, p21 and plasminogen activator inhibitor‐1 (PAI‐1) expression, and caused cell death without affecting SASP in hyperoxia‐induced senescent fASM. MitoQ prevented increases in senescence markers and SASP in hyperoxia‐exposed fASM. These findings demonstrate distinct and viable strategies to counter hyperoxia‐induced fASM senescence. Fucoidan (senomorphic), D + Q (senolytic), and MitoQ (prophylactic antioxidant) represent promising, mechanistically different approaches to alleviate detrimental effects of moderate hyperoxia in developing airways towards limiting perinatal lung disease.

EndMT has been reported to contribute to the pathogenesis of PAH. In this study, we aimed to determine the role of Ca 2+ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β 1 requires store-operated Ca 2+ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time, we demonstrated that TGF-β 1 -induced EndMT is a Ca 2+ -dependent event, whereas inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β 1 .

Abstract Nicotine exposure in the context of smoking or vaping worsens airway function. Although nicotinic acetylcholine receptors (nAChRs) are commonly thought to exert effects through the peripheral nervous system, we previously showed that airway smooth muscle (ASM) expresses them, particularly α7 subtype nAChR (α7nAChR), with functional effects on contractility and metabolism. However, the mechanisms of nAChR regulation and downstream effects in ASM are not fully understood. Using ASM cells from people without asthma versus people with mild to moderate asthma, we tested the hypothesis that nAChR-specific endoplasmic reticulum (ER) chaperones, resistance to inhibitors of cholinesterase 3 (RIC-3) and transmembrane protein 35A (TMEM35A) promote cell surface localization of α7nAChR with downstream influence on its functionality: effects exacerbated by inflammation. We found that mild to moderate asthma and exposure to proinflammatory cytokines relevant to asthma promote chaperone and α7nAChR expression in ASM. Downstream, ER stress was linked to nicotine/α7nAChR signaling, where RIC-3 and TMEM35 regulate nicotine-induced ER stress, intracellular Ca2+ regulation, and ASM cell proliferation. Overall, our data highlight the importance α7nAChR chaperones in mediating and modulating nicotine effects in ASM toward airway contractility and remodeling.

Background: Limited information is available for patients with breast cancer (BC) and coronavirus disease 2019 (COVID-19), especially among underrepresented racial/ethnic populations. Methods: This is a COVID-19 and Cancer Consortium (CCC19) registry-based retrospective cohort study of females with active or history of BC and laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection diagnosed between March 2020 and June 2021 in the US. Primary outcome was COVID-19 severity measured on a five-level ordinal scale, including none of the following complications, hospitalization, intensive care unit admission, mechanical ventilation, and all-cause mortality. Multivariable ordinal logistic regression model identified characteristics associated with COVID-19 severity. Results: 1383 female patient records with BC and COVID-19 were included in the analysis, the median age was 61 years, and median follow-up was 90 days. Multivariable analysis revealed higher odds of COVID-19 severity for older age (aOR per decade, 1.48 [95% CI, 1.32–1.67]); Black patients (aOR 1.74; 95 CI 1.24–2.45), Asian Americans and Pacific Islander patients (aOR 3.40; 95 CI 1.70–6.79) and Other (aOR 2.97; 95 CI 1.71–5.17) racial/ethnic groups; worse ECOG performance status (ECOG PS ≥2: aOR, 7.78 [95% CI, 4.83–12.5]); pre-existing cardiovascular (aOR, 2.26 [95% CI, 1.63–3.15])/pulmonary comorbidities (aOR, 1.65 [95% CI, 1.20–2.29]); diabetes mellitus (aOR, 2.25 [95% CI, 1.66–3.04]); and active and progressing cancer (aOR, 12.5 [95% CI, 6.89–22.6]). Hispanic ethnicity, timing, and type of anti-cancer therapy modalities were not significantly associated with worse COVID-19 outcomes. The total all-cause mortality and hospitalization rate for the entire cohort was 9% and 37%, respectively however, it varied according to the BC disease status. Conclusions: Using one of the largest registries on cancer and COVID-19, we identified patient and BC-related factors associated with worse COVID-19 outcomes. After adjusting for baseline characteristics, underrepresented racial/ethnic patients experienced worse outcomes compared to non-Hispanic White patients. Funding: This study was partly supported by National Cancer Institute grant number P30 CA068485 to Tianyi Sun, Sanjay Mishra, Benjamin French, Jeremy L Warner; P30-CA046592 to Christopher R Friese; P30 CA023100 for Rana R McKay; P30-CA054174 for Pankil K Shah and Dimpy P Shah; KL2 TR002646 for Pankil Shah and the American Cancer Society and Hope Foundation for Cancer Research (MRSG-16-152-01-CCE) and P30-CA054174 for Dimpy P Shah. REDCap is developed and supported by Vanderbilt Institute for Clinical and Translational Research grant support (UL1 TR000445 from NCATS/NIH). The funding sources had no role in the writing of the manuscript or the decision to submit it for publication. Clinical trial number: CCC19 registry is registered on ClinicalTrials.gov, NCT04354701 .

Asthma is a disease characterized by airway hyperresponsiveness and inflammation. One‐third of pregnant women with pre‐existing disease experience a decrease in symptoms during the third trimester, when estrogen levels are highest. Previous studies have shown that estrogens relax vascular smooth muscle, however, little is known about estrogen effects on airway smooth muscle (ASM). We tested the hypothesis that estrogen modulates human ASM contractility. Western blot analysis and immunohistochemical studies showed the presence of both full‐length and truncated isoforms of ERα and β. Furthermore, ER isoforms were localized to caveolae. Caveolin‐1 siRNA downregulated ERα isoform in human ASM. Exposure to the pro‐inflammatory cytokine TNFα substantially increased ERα expression in ASM. In fura‐2 loaded human ASM cells, acute exposure to biologically‐active 17β‐estradiol decreased the intracellular Ca 2+ response to contractile agonists such as histamine and bradykinin; this effect was blunted by the ER antagonist ICI 182, 780. Overall, these data suggest that estrogens may have a bronchodilatory effect. Accordingly, increased ER expression with airway inflammation may actually be protective during pregnancy in that bronchodilation resulting from higher estrogen levels (acting via ERs) may alleviate the increased ASM contractility of asthma. Supported by the Mayo Graduate School and NIH grant UL1RR024150‐01(Mayo Clinic CTSA awards to YSP and CMP)

Store operated calcium entry (SOCE) is important for regulation of intracellular Ca 2+ ([Ca 2+ ] i ) in neurons. Recent studies in other cell type suggest that stromal interaction molecule (STIM1) triggers SOCE. We examined the role of STIM1 in the sensitivity of SOCE to temperature, pH and glutamate in differentiated, fura‐2 loaded rat pheochromocytoma (PC12) cells, a model for neuronal cells. In STIM1 siRNA‐transfected vs. control cells, SOCE was triggered by introduction of extracellular Ca 2+ following endoplasmic reticulum depletion via cyclopiazonic acid in zero Ca 2+ . Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40 o C) or different concentrations of glutamate. In controls, SOCE was progressively inhibited by decreased pH or temperature, while alkalosis and increased temperature stimulated Ca 2+ influx. Increasing glutamate concentrations progressively inhibited SOCE. siRNA inhibition of STIM1 substantially blunted both temperature sensitivity (especially increased temperature) and pH sensitivity (especially acidic pH) of SOCE. These data show that SOCE in PC12 cells is susceptible to acidosis, temperature, and agonist stimulation: factors that can adversely affect neuronal function under pathological conditions. In this regard, the SOCE triggering protein STIM1 may play a role in such susceptibility. This work has been supported by NIH grants UL1RR024150‐01(CR to CMP, CR20 and Early Career Development Award to YSP), and the Department of Anesthesiology (CMP, YSP), Mayo Clinic College of Medicine, Rochester, MN

Intracellular Ca 2+ regulation in human airway smooth muscle (hASM) involves plasma membrane (PM) Ca 2+ influx, including that triggered by sarcoplasmic reticulum (SR) Ca 2+ depletion, i.e. store‐operated Ca 2+ entry (SOCE). While transient receptor potential channels (e.g. TRPC3, TRPC4) are thought to mediate SOCE, the mechanisms triggering or regulating SOCE are being actively investigated. Recent studies suggest that stromal interaction molecule 1 (STIM1) acts as a signaling mediator between PM and SR, potentially regulating Ca 2+ entry via SOCE. In hASM, fluorescence microscopy and western blot analyses were used to verify the presence of STIM1. SR Ca 2+ depletion with 10μM cyclopiazonic acid (CPA) resulted in increased STIM1 expression in the PM fraction, compared to controls. Transfection with 50nM STIM1 siRNA for 48h significantly decreased STIM1 levels. In cells where [Ca 2+ ] i was measured using fura 2‐AM, SOCE was significantly reduced in cells transfected with STIM1 siRNA. Green‐fluorescent protein (GFP)‐tagged STIM1 transfection showed intracellular localization in non‐stimulated cells, with increased PM staining following CPA exposure. Subsequent immunostaining revealed co‐localization of STIM1 with TRPC3 and TRPC4. These results indicate that STIM1 is present in hASM and suggest a role for this protein in regulating SOCE. This work has been supported by NIH grants UL1RR024150‐01(CR to CMP, CR20 and Early Career Development Award to YSP), HL74309 (GCS), and the Department of Anesthesiology (CMP, YSP), Mayo Clinic College of Medicine, Rochester, MN

Smoking contributes to or exacerbates diseases such as asthma and COPD, where airway hyperreactivity is a key feature. Enhanced Ca 2+ in airway smooth muscle contributes to airway hyperreactivity. We previously determined that the cytokine TNFα upregulates expression of Ca 2+ regulatory molecules such as membrane bound bifunctional enzyme CD38 and Ca 2+ influx regulatory TRPC3 in human airway smooth muscle cells (HASM). We hypothesized that cigarette smoke (CS) is an inflammatory stimulus that upregulates Ca 2+ regulatory mechanisms in HASM. Western analysis showed increased expression of CD38, TRPC3 and TRPC6 in lungs from mice following 4 week in vivo CS exposure. These proteins were also upregulated in HASM cells incubated in 1 or 2% CS extract (CSE), as well as in HASM samples from patients with COPD (compared to controls). In fura‐2 loaded HASM cells, exposure to CSE enhanced histamine‐mediated Ca 2+ mobilization as well as store‐operated calcium entry: effects substantially blunted by TRPC3 siRNAs. Finally, in living lung slices of mice exposed for 4 weeks to CS, ACh‐induced airway contractility was substantially greater, compared to controls. These results indicate that CS upregulates expression and function of Ca 2+ regulatory proteins, thus contributing to airway hyperreactivity in smoking related diseases. Supported by the Annenberg Foundation (MEW), HL090595 (CMP), FAMRI (RV, YSP).

Supplemental oxygen places the immature airway at risk for bronchopulmonary dysplasia and asthma. Hyperoxia enhances intracellular Ca2+ ([Ca2+]i) and contractility of airway smooth muscle (ASM), and impairs relaxation (potentially by altering cyclic nucleotide levels). The neurotrophin brain‐derived neurotrophic factor (BDNF) enhances [Ca2+]i in adult ASM. We tested the hypothesis that hyperoxia‐induced airway hyperreactivity involves cyclic nucleotide‐mediated enhancement of BDNF release in human fetal (18–22 weeks) ASM cells exposed to 21% (normoxia) or 80% oxygen in the presence/absence of 1mM dibutyryl cAMP (DBcAMP) or 8‐Br‐cGMP. BDNF release (ELISA) was increased by hyperoxia (143% of normoxia). DBcAMP enhanced BDNF release with normoxia (242% of baseline) and even more with hyperoxia (280%), while 8‐Br‐cGMP blunted BDNF release in normoxia (57% of baseline) but to a lesser extent with hyperoxia (68% of baseline). Measurements of [Ca2+]i in fetal ASM cells showed DBcAMP increasing [Ca2+]i responses to histamine or ACh (effects enhanced with hyperoxia). Further, chelation of released BDNF using chimeric TrkB‐Fc (1?g/ml) reversed DBcAMP effects and blunted [Ca2+]i responses to below controls. We conclude that an imbalance between cAMP vs. cGMP enhances BDNF release in the developing airway, resulting in greater airway reactivity with hyperoxia. Supported by NIH grants HL056470 and HL088029

Women have a higher incidence and severity of asthma exacerbations compared to men. Asthma symptoms fluctuate with hormonal status suggesting a role for sex steroids in modulating bronchoconstriction. We have previously shown that acute estrogen decreases intracellular calcium in human airway smooth muscle thereby facilitating bronchodilation, similar to estrogen‐induced vasodilation. Akin to endothelium, airway epithelium is a source of nitric oxide (NO), a bronchodilator. We hypothesized that estrogens facilitate bronchodilation by generating NO in bronchial epithelium. In acutely dissociated human bronchial epithelial cells, 5–10 min exposure to 1 μM ACh or 17β‐estradiol (E2) (1 and 10 nM) resulted in increased DAF‐2 fluorescence (NO indicator). Both ER specific agonists THC (ERα) and DPN (ERβ) (10 nM,) induced NO production comparably. E2 effects were abrogated by the estrogen receptor (ER) antagonist ICI 182,780 (Faslodex®). eNOS phosphorylation was enhanced by E2 while E2‐induced NO production was attenuated by Ca2+ chelation (BAPTA; 20 μM, 30 min) and eNOS inhibition (L‐NAME). Overall, these data indicate a role for airway epithelium‐derived NO in the bronchodilatory effects of estrogens in human ASM. Supported by NIH grants HL090595 (CMP), HL088029 & HL56470 (YSP), and Mayo Graduate School (EAT).