Thompson, Michael D.

Abstract Rationale: Moderate hyperoxia (<60% O2) in premature infants promotes bronchial airway hyperresponsiveness (AHR) and impaired bronchodilation involving airway smooth muscle (ASM). Understanding mechanisms by which hyperoxia initiates long-term bronchial airway changes in prematurity is critical for novel therapies for wheezing disorders and asthma in children. Underdeveloped lungs in prematurity have immature antioxidant systems, which may be due to disrupted dynamics that promote susceptibility to oxidative stress from O2. Cytoplasmic and mitochondrial calcium signaling mechanisms and O2 metabolism are pathways highly relevant to ASM and airway disease. We propose that the novel gasotransmitter hydrogen sulfide (H2S) attenuates effects of O2 in developing ASM and hypothesize that hyperoxia in prematurity promotes cellular and mitochondrial calcium signaling dysfunction, effects which are counteracted by H2S.Methods: Human fetal (fASM) cells isolated from tracheobronchial samples of de-identified 18-22 week fetuses following fetal demise (Mayo IRB approved as exempt) were exposed to 21% vs 40% O2. fASM were treated 24-72 hours with H2S donor GYY4137, mitochondrial-specific H2S donor AP39, cystathione-β-synthase (CBS) stabilizer AdoMet, cAMP inhibitors SQ22536 or KT5720, or vehicle control (DMSO). CBS shRNA lentiviral particles were used to generate stable knockdown cell lines. Protein markers were measured by Western blot. Intracellular calcium ([Ca2+]i) was measured using 5μM Fura-2/AM and mitochondrial calcium ([Ca2+]m) with 1μM Rhod-2 AM using fluorescence microscopy. Results: CBS expression was lower in fASM exposed to 40% O2 compared to 21% O2. Lentivirus induced CBS KD was exacerbated by 40% O2. Hyperoxia increased [Ca2+]i and [Ca2+]m response to histamine (10μM) in vehicle and Scr controls compared to normoxia. CBS KD increased [Ca2+]i and [Ca2+]m responses to histamine compared to Scr fASM in 21% O2. CBS KD fASM exposed to hyperoxia showed increased [Ca2+]i and [Ca2+]m responses to histamine, but the effect was not amplified by CBS KD. [Ca2+]i and [Ca2+]m responses to histamine were decreased in normoxia by H2S donors GYY4137 (50μM) and AP39 (100nM) and CBS stabilizer AdoMet (100nM); effects which also blunted hyperoxia induced increases. SQ22536 and KT5720 inhibited effects of AP39 and GYY4137 in hyperoxia. Conclusions:Our findings suggest hyperoxia leads to [Ca2+]i and [Ca2+]m dysfunction in developing airway cells, which can be alleviated through modulation of H2S pathways, offering potential targets for future therapies to blunt hyperoxia effects on bronchoconstriction and bronchodilation. Acknowledgment: Supported by AHA Postdoctoral Fellowship 20POST35210002 (Bartman), NIH R01 HL-056470 (Prakash), and HL-160570 (Pabelick).

Neurotrophins are growth factors traditionally associated with neuronal structure and function; however, neurotrophins and their receptors have been found throughout the lung, including airway, pulmonary artery and endothelium although their roles within the lung are still under investigation. We tested the hypothesis that brain derived neurotrophic factor (BDNF) enhances pulmonary artery constriction. In endothelium‐intact arterial rings, BDNF (acute 10nM, 1h; chronic 1nM, 18h) did not affect phenylephrine‐induced contraction, but enhanced subsequent ACh‐induced relaxation. These effects were blunted by endothelium‐denudation. In fura‐2 loaded human pulmonary artery smooth muscle cells, acute and chronic BDNF decreased the Ca 2+ responses to 10 μM bradykinin. In human pulmonary artery exposed to TNFα (50 ng/mL, 18h), BDNF levels were decreased, compared to human airway, where previous studies under identical treatment resulted in increased levels. These data refute our hypothesis and suggest that neurotrophins, working via endothelial cells, enhance vasodilation in the pulmonary vasculature. Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.

Airway inflammation leads to increased intracellular Ca 2+ ([Ca 2+ ] i ) in airway smooth muscle (ASM). There is now growing evidence that the bidrectional plasma membrane Na + /Ca 2+ exchange (NCX) may contribute to ASM [Ca 2+ ] i regulation. However, the roles of NCX‐mediated Ca 2+ influx vs. efflux, or the role of NCX in airway inflammation are largely unknown. In the present study, we examined NCX expression and function in human ASM exposed to the pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis of human ASM tissue showed significant expression of the NCX1 isoform, with substantial increase in expression following overnight cytokine exposure. In fura‐2 loaded human ASM cells, NCX‐mediated Ca 2+ influx as well as efflux (measured as slopes of changes in [Ca 2+ ] i ) was elicited using techniques previously described for cardiac muscle. Contribution of NCX was verified by measuring [Na + ] i using the fluorescent Na + indicator SBFI. NCX‐mediated influx was verified by demonstrating prevention of rising [Ca 2+ ] i or falling [Na + ] i in the presence of the NCX inhibitor KBR7943. In second set of studies, NCX siRNA transfection of human ASM substantially blunted efflux and influx mode of NCX (siRNA effect verified by Western blot). Exposure to TNF‐α as well as IL‐13 significantly enhanced influx mode of NCX. Although both cytokines also increased efflux mode of NCX, these effects were relatively smaller. These data suggest that NCX may be one mechanism contributing to inflammation‐induced enhancement of [Ca 2+ ] i. Supported by NIH grants HL74309, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

The mechanisms underlying pulmonary hypertension are still being explored. Recent studies suggest a role for caveolae (pit‐like plasma membrane structures), although it is not entirely clear whether they enhance or blunt pulmonary hypertension. The proteins caveolins ‐1, and ‐2, and polymerase I and transcription release factor (PTRF) are essential to forming caveolae. In other tissues, growth factors called neurotrophins (e.g. brain‐derived neurotrophic factor, BDNF) are thought to affect cell proliferation and structure. This study investigated caveolins and neurotrophins in human pulmonary artery. Western analysis of artery from a patient with primary pulmonary hypertension showed decreased levels of caveolin‐1, caveolin‐2, PTRF and RhoA. Exposure of human pulmonary artery to 1 nM BDNF increased protein levels of caveolin‐1 and PTRF. Isolated pulmonary artery smooth muscle cells exposed to BDNF showed decreased [Ca 2+ ] i response to bradykinin (10 nM) and histamine (10 μM), but no change in caveolin or PTRF levels. These data suggest that decreased caveolin levels may contribute to increased pulmonary vascular contractility, while neurotrophins (potentially by increasing caveolin levels) may alleviate excessive constriction. Further studies are required to determine whether neurotrophins affect caveolae in endothelial cells or smooth muscle. Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.

Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are important mechanisms in airway smooth muscle (ASM) intracellular Ca 2+ [Ca 2+ ] i regulation. In other tissues, there is increasing evidence for a role of mitochondria in [Ca 2+ ] i buffering, especially following SR Ca 2+ release. Mitochondrial buffering may also influence Ca 2+ influx. The purpose of the present study was to examine the role of mitochondria in [Ca 2+ ] i buffering in human ASM. Cells were loaded with fura‐2 and exposed to 10 µM histamine in "0" Ca 2+ HBSS containing 1mM lanthanum and 1µM nifedipine (to block Ca 2+ influx/efflux) and in the additional presence of 1µM RU360 (mitochondrial Ca 2+ uniporter inhibitor) or 1µM CGP37157 (CGP; mitochondrial Na + /Ca 2+ exchange inhibitor). Compared to controls, peak [Ca 2+ ] i levels were increased by 30% in the presence of CGP, while RU360 had not effect. Both CGP and RU360 significantly increased store‐operated Ca 2+ entry. In cells co‐loaded with fura‐2 and rhod‐2 (mitochondrial Ca 2+ indicator), exposure to 5 µM caffeine showed instantaneous SR Ca 2+ release followed by an increase in [Ca 2+ ] mito . Exposure to 1 µM ACh resulted in ACh‐induced [Ca 2+ ] i oscillations that were reflected by mitochondria (but with a time delay). In ASM cells co‐labeled for mitochondria (MitoTracker) and SR (fluorescent ryanodine) migration of mitochondria towards SR was observed upon exposure to ACh or cyclopiazonic acid. This migration was completely inhibited by phalloidin (actin inhibitor). These data demonstrate an important role of mitochondria in [Ca 2+ ] i buffering in ASM. Supported by NIH grant RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.

Intracellular Ca 2+ ([Ca 2+ ] i ) levels are increased in inflamed airway smooth muscle (ASM). Sarcoplasmic reticulum (SR) Ca 2+ release and reuptake are key components of ASM [Ca 2+ ] i regulation. Ca 2+ reuptake occurs via the ATPase SERCA. We hypothesized that inflammation disrupts SERCA function, contributing to increased [Ca 2+ ] i . We examined SERCA expression and regulation in human ASM exposed to pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis showed significantly decreased SERCA expression following overnight cytokine exposure. In other tissues, SERCA is regulated by the inhibitory protein phospholamban (PLB). We found that human ASM does not express PLB protein (although mRNA is detectable). Cytokine exposure did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (potentially via CaMKII) occurs in human ASM. In fura‐2 loaded human ASM cells, we examined [Ca 2+ ] i responses to ACh or bradykinin (zero extracellular Ca 2+ ) and found significant reduction in rates of fall of [Ca 2+ ] i transients in cells exposed to TNF‐α or IL‐13. We further found that CaMKII antagonist KN‐93 significantly slowed the fall of [Ca 2+ ] i transients in both controls and cytokine‐exposed human ASM cells. These data suggest that a) SERCA in human ASM is regulated by mechanisms such as CaMKII; and b) airway inflammation decreases SR refilling by blunting SERCA expression and function. Supported by NIH grants HL74309, the Flight Attendants Medical Research Institute (FAMRI), and the Department of Anesthesiology, Mayo Clinic.

Background Uterine leiomyomas (fibroids) are benign smooth muscle cell tumors of the uterus that affect a large percentage of reproductive aged women, contribute to infertility, excessive menstrual bleeding, and represent the leading cause of hysterectomy (~70%). Despite their importance, the mechanisms that promote fibroid growth are still not well‐understood. Fibroids are comprised of smooth muscle cells (SMCs) and fibroblasts and their growth is modulated by hormones including estrogen and progesterone. Intracellular calcium [Ca 2+ ] i signaling is a key aspect of SMC physiology in promoting contractility, cell proliferation, and fibrosis: aspects relevant to fibroid biology. While [Ca 2+ ] i regulation in normal myometrium is well characterized, this has not been explored in fibroids. The goal of this study was to explore [Ca 2+ ] i regulation in fibroid SMCs and the effect of sex steroids towards obtaining insights into tumor progression. Methods Cells were enzymatically isolated from normal myometrium and select fibroids of different sizes from patients undergoing myomectomy at Mayo Clinic, Rochester, MN (IRB approved). [Ca 2+ ] i was measured using fura‐2/AM and real‐time fluorescence microscopy. The contribution of Ca 2+ influx vs. intracellular pathways in the responses to 1uM oxytocin were assessed using pharmacological inhibitors/activators. Cells were pretreated with vehicle, 1nM estrogen, or 1nM progesterone to assess the effect of sex steroids. Results Fibroid‐derived cells showed a greater contribution of Ca 2+ influx pathways in their responses to oxytocin than normal myometrium. Here, the Ca 2+ ‐activated chloride channel TMEM16A (Ano1) was found to be contributory, with Western analysis showing higher levels of Ano1 in fibroids. Exposure to estrogen resulted in significantly higher [Ca 2+ ] i responses compared to cells treated with vehicle or progesterone, although progesterone did enhance [Ca 2+ ] i responses as well. Estrogen increased Ano1 expression. Cell proliferation was also increased in fibroid cells. Conclusions Exposure to estrogen and progesterone enhances oxytocin induced [Ca 2+ ] i responses of fibroid‐derived cells, potentially via increased TMEM16A (Ano1). Such increases may further contribute to the observed hormone‐induced enhancement of fibroid cell proliferation. Menstrual cycle fluctuations in reproductive aged women may thus contribute to the progression of fibroids. Greater understanding of calcium signaling pathways such as Ano1 in fibroid tissue may allow for novel therapeutic targets in uterine leiomyomas. Support or Funding Information Supported by NIH grants R25 GMO75148 (JLR), R01 HL088029 (YSP), and R01 HL56470 (YSP). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .