Ravix, Jovanka

Background Uterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in >25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth. Methods Cells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively. Results Significant increases in transcripts for ECM gene Collagen 1 ( COL1A1 ) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor ( CTGF ), alpha smooth muscle actin ( ACTA2 ), Endothelin 1 ( EDN1 ), and plasminogen activator inhibitor 1 ( SERPINE1 ) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 ( PTGS2 ) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium. Conclusions Based on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth. Support or Funding Information None This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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 .

Abstract Airway smooth muscle (ASM) regulation of airway structure and contractility is critical in fetal/neonatal physiology in health and disease. Fetal lungs experience higher Ca 2+ environment that may impact extracellular Ca 2+ ([Ca 2+ ] o ) sensing receptor (CaSR). Well‐known in the parathyroid gland, CaSR is also expressed in late embryonic lung mesenchyme. Using cells from 18‐22 week human fetal lungs, we tested the hypothesis that CaSR regulates intracellular Ca 2+ ([Ca 2+ ] i ) in fetal ASM (fASM). Compared with adult ASM, CaSR expression was higher in fASM, while fluorescence Ca 2+ imaging showed that [Ca 2+ ] i was more sensitive to altered [Ca 2+ ] o . The fASM [Ca 2+ ] i responses to histamine were also more sensitive to [Ca 2+ ] o (0–2 mM) compared with an adult, enhanced by calcimimetic R568 but blunted by calcilytic NPS2143. [Ca 2+ ] i was enhanced by endogenous CaSR agonist spermine (again higher sensitivity compared with adult). Inhibition of phospholipase C (U73122; siRNA) or inositol 1,4,5‐triphosphate receptor (Xestospongin C) blunted [Ca 2+ ] o sensitivity and R568 effects. NPS2143 potentiated U73122 effects. Store‐operated Ca 2+ entry was potentiated by R568. Traction force microscopy showed responsiveness of fASM cellular contractility to [Ca 2+ ] o and NPS2143. Separately, fASM proliferation showed sensitivity to [Ca 2+ ] o and NPS2143. These results demonstrate functional CaSR in developing ASM that modulates airway contractility and proliferation.

Abstract Supplemental O2 (hyperoxia; 30–90% O2) is a necessary intervention for premature infants, but it contributes to development of neonatal and pediatric asthma, necessitating better understanding of contributory mechanisms in hyperoxia-induced changes to airway structure and function. In adults, environmental stressors promote formation of senescent cells that secrete factors (senescence-associated secretory phenotype), which can be inflammatory and have paracrine effects that enhance chronic lung diseases. Hyperoxia-induced changes in airway structure and function are mediated in part by effects on airway smooth muscle (ASM). In the present study, using human fetal ASM cells as a model of prematurity, we ascertained the effects of clinically relevant moderate hyperoxia (40% O2) on cellular senescence. Fetal ASM exposed to 40% O2 for 7 days exhibited elevated concentrations of senescence-associated markers, including β-galactosidase; cell cycle checkpoint proteins p16, p21, and p-p53; and the DNA damage marker p-γH2A.X (phosphorylated γ-histone family member X). The combination of dasatinib and quercetin, compounds known to eliminate senescent cells (senolytics), reduced the number of hyperoxia-exposed β-galactosidase–, p21-, p16-, and p-γH2A.X–positive ASM cells. The senescence-associated secretory phenotype profile of hyperoxia-exposed cells included both profibrotic and proinflammatory mediators. Naive ASM exposed to media from hyperoxia-exposed senescent cells exhibited increased collagen and fibronectin and higher contractility. Our data show that induction of cellular senescence by hyperoxia leads to secretion of inflammatory factors and has a functional effect on naive ASM. Cellular senescence in the airway may thus contribute to pediatric airway disease in the context of sequelae of preterm birth.