Prakash, Y.S.

Abstract Rationale: In premature infants, continuous positive airway pressure (CPAP) serves as an effective respiratory support strategy in the NICU. However, the static stretch induced by CPAP may negatively impact bronchial airway development, leading to hyperreactivity and remodeling, key proponents of asthma. Here, epithelial and airway smooth muscle cells are affected by stretch, but the mechanisms by which effects occur are not well-known. In airway smooth muscle contractility, stromal interaction molecule 1 (STIM1) at the endoplasmic reticulum and plasma membrane Orai1 channels are important mediators of the store operated calcium entry (SOCE) microdomain signaling pathway, facilitating movement of calcium into the cell and triggering downstream remodeling effects. Recently discovered mechanosensitive Piezo (PZ) channels play a critical role in calcium influx upon mechanical stimulation. In this study, we investigated the hypothesis that interplay between mechanosensitive channels such as PZ and the SOCE microdomain promotes calcium in human fetal airway smooth muscle cells (fASM) towards increasing contractility by stretch. Methods: fASM were obtained from 18-22 week abortus following fetal demise (exempt per Mayo IRB). Cells were pretreated with either PZ1 inhibitor GsMTx4 [1uM] or PZ2 inhibitor D-GsMTx4 [1uM] 45 minutes before stretch. Cells were exposed to normal breathing stretch conditions on a FlexCell System at a 5% cyclic strain (40 cycles/minute) and CPAP with static stretch superimposed for 24 hours. Protein expression of PZ channels and key SOCE proteins were quantified. SOCE responses of stretched and control cells were determined using established protocols in fura-2 loaded cells using fluorescence microscopy. Results: Mechanical stretching of fASM increased protein expression of PZ1, STIM1, and Orai1 in a dose dependent manner. Stretch increased intracellular SOCE responses compared to non-stretched controls. PZ1 antagonist GsMTx4 and PZ2 antagonist D-GsMTx4 inhibited stretch-induced Piezo1, STIM1, and Orai1 upregulation. GsMTx4 and D-GsMTx4 blunted stretch and non-stretched induced calcium SOCE responses relative to unstretched control. Conclusions: Our findings suggest that in the context of mechanical stretch, Piezo channels and the downstream SOCE signaling pathway increase in expression and functionality in the fetal airway which may contribute to CPAP associated changes in airway contractility.

Abstract Rationale: Within the asthmatic lung, inflammatory signaling alters the contractile capacity of airway smooth muscle (ASM). In chronic inflammation, ASM become hypercontractile and are a key contributor toward airway hyperresponsiveness (AHR). As a heterogenous disorder, several asthmatic endotypes have been defined. A severe ASM endotype is partly defined as being glucocorticoid insensitive. We have reported the role of Kisspeptin (Kp)/KISS1R signaling in the lung in the context of airway fibrosis. In this study, KISS1R and Kp-10 were investigated for a potential interactions with intracellular calcium [Ca2+]i signaling and ASM contractility in the in vitro setting of severe asthmatic endotypes. Methods: Primary ASM were obtained from non-pathological samples from the Mayo Clinic. ASM cells were treated with inflammatory cytokines (TNFα 20 ng/ml, or IL-13 50 ng/mL)± Kp-10 (1 μM). The steroid-insensitive asthma endotype was investigated using pro-inflammatory cytokines (TNFα 20 ng/ml and IFN-γ 25 ng/mL) ± a corticosteroid (Fluticasone Propionate 100 nM). Β2-Adrenergic Receptor (β2-AR) desensitized endotypes were developed by treating cells with a β2-AR agonist (Salbutamol 1 μM) for 24 hours prior to and along with cytokine treatments (TNFα 20 ng/ml and IFN-γ 25 ng/mL or IL-13 50 ng/mL) ± Kp-10. [Ca2+]i was measured via Fura-2AM (4 μM) labeling on an Olympus Fluoview 300 microscope. Myosin light chain (MLC) phosphorylation studies were used to quantify downstream mechanics of ASM contractility. Results: KISS1R activation in IL-13 treated cells reduced [Ca2+]i in response to histamine. In contrast, TNFα treated cells were responsive to Kp-10 for acute treatments, however this effect was lost after 24 hours of Kp-10 treatment. TNFα and IFN-γ treated cells showed a robust [Ca2+]i response to histamine, and both fluticasone (∼20% reduction) and Kp-10 (∼10% reduction) alone had a minimal effect in reducing [Ca2+]i. Fluticasone and Kp-10 co-exposure significantly reduced [Ca2+]i and significantly reduced the effect of TNFα/IFN-γ (∼45% reduction). In B2-AR desensitized cells, Kp-10 exposure maintained the effect of reducing [Ca2+]i, suggesting Kp-10/KISS1R signaling works through an alternate or independent pathway. Kp-10 treatments on cytokine exposed cells reduced p-MLC in response to histamine, suggesting a role in ASM contractility. Conclusion: The increase of [Ca2+]i in inflamed cells is associated with AHR; Kp-10/KISS1R signaling showed varying efficacy in reducing [Ca2+]i within inflammatory endotypes. These results also highlight that Kp-10 acts synergistically with the current therapeutics available for asthma.