Pabelick, Christina M.

Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.

Chronic airway disease is a short- and long-term consequence of premature birth. Understanding effects of O2 exposure during the perinatal period is key to identify targetable mechanisms that initiate and sustain adverse airway changes. Our findings show a beneficial effect of exogenous H2S on developing mouse airway structure and function with notable sex differences. H2S donors alleviate effects of O2 on airway hyperreactivity, contractility, airway smooth muscle thickness, and extracellular matrix deposition.

Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Piezo channels were first described just over a decade ago and their function in the lung is largely unknown. We found that piezo channels are present and functional in the developing airway and contribute to intracellular calcium responses and extracellular matrix remodeling in the setting of stretch. This may improve our understanding of the mechanisms behind development of chronic airway diseases, such as asthma, in former preterm infants exposed to respiratory support, such as continuous positive airway pressure (CPAP).

Rationale Human chromosomal anomalies, notably trisomies, induce several alterations in gene expression, leading to various disease phenotypes. Recent studies showed that dermal fibroblasts from patients with trisomy 13 (T13), T18 or T21 present features of increased senescence and oxidative stress. We recently demonstrated that T21 lungs present abnormalities prenatally. Senescence is a state of cell division arrest that plays an important role in organogenesis. However, little is known about cellular senescence in fetal human lung with T13, T18 and T21. We therefore sought to characterize cellular senescence in these lung tissues. Methods Fresh human fetal lung tissues with T13, T18 and T21 were collected along with euploid age and sex matched controls. RT-qPCR and immunofluorescence staining (IF) were performed to assess the expression of senescence markers as well as Senescence-Associated Secretory Phenotype (SASP) elements in the various lung compartments (epithelial and mesenchymal). In addition, fibroblasts were isolated and cultured from each sample to determine the extent of senescence on isolated mesenchyme by IF and RT-qPCR. Staining for oxidative stress detection and Reactive oxygen species were assessed by CellROX and MitoSOX. Results RT-qPCR analysis in T13 whole lung tissues demonstrated no significant differences in the expression of either senescence or SASP markers as compared to control ( P16/ P21/ TP53 and IL6/ IL1 β/ TGF β/ CXCL8 respectively; n=5). Interestingly however, IF staining in T13 tissues showed a significant increase of γ-H2AFX, a marker of senescence, in the mesenchyme (n=4) as well as significantly decreased P21 staining in both the epithelial and mesenchymal compartments. Additionally, isolated fibroblasts from T13 lungs expressed significantly more IL6 as compared to control (n=3), with IF staining demonstrating a significant increase of γ-H2AFX. T18 lungs displayed significantly increased expression of P16 as compared to control (n=6), whereas IF staining revealed an increase of γ-H2AFX in whole tissue (n=4). Fibroblasts isolated from T18 lungs revealed no significant difference in any markers (gene or protein). Lastly, T21 lungs showed a significant increase of CXCL8 expression as compared to control (n=6). IF staining of T21 lungs demonstrated an increase γ-H2AFX in both the epithelium and mesenchyme (n=8). However, isolated T21 lung fibroblasts displayed significantly increased expression of P16, P21, TP53, and CXCL8. Furthermore, IF staining of T21 lung fibroblasts displayed significantly more γ-H2AFX and P21 positive cells as compared to controls (n=5). Interestingly, only cells from T21 lungs presented a significant increase of CellROX and MitoSOX staining (n=5). Conclusion In this study we established that expression of both senescence and SASP markers differ depending on the trisomy. Our results suggest that cellular senescence is more pronounced in lungs from individuals with trisomy 21. These authors acknowledge funding from NIH/NHLBI R01HL141856 (to DAA); NIH/ Office Of The Director, National Institutes Of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DAA); CIRM training grant EDUC4-12837 (RB) and NIH/NICHD 2R24HD000836-52 (IG). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Multiple asthma phenotypes are driven by pathobiological heterogeneity. Almost 25% of asthma patients do not respond well to current treatments like inhaled corticosteroids, urging researchers and clinicians to propose novel treatment targets and strategies. Orphan type C G-protein coupled receptor GPRC5B is one such novel target abundantly expressed in various tissues, but there are no known ligands for GPRC5B. Previously, we demonstrated that orphan type C G-protein coupled receptor GPRC5B is increasingly expressed in human asthmatic airways and plays a crucial role in regulating intracellular calcium ([Ca2+]i) in human airway smooth muscle (ASM) cells. However, the role of GPRC5B activation on airway contraction is not identified yet. Our objective for this study is to design and synthesize a novel ligand for GPRC5B and determine the effect of GPRC5B activation on [Ca2+]i responses in ASM cells and airway contractility in ex vivo murine airways. Methods: ZincPharmer was used for pharmacophore search. After energy minimization, the top hit compound was synthesized using a standard amide coupling reaction, purified by flash chromatography, and characterized by NMR spectroscopy. Human ASM cells loaded with Fura-2 AM were imaged for [Ca2+]i responses. Silencer select siRNA was used for transient silencing of GPRC5B in ASM cells. Myosin light chain (MLC) phosphorylation was measured using immunoblot analysis. Precision-cut lung slices (PCLS) (~200 μm thickness) of wild-type C57BL6 mice were used for contractile studies. Results: Based on molecular docking data, the top compound N-(5-chloro-2-hydroxybenzyl)-2-((tetrahydro-2H-pyran-4-yl)oxy) acetamide (L1) was synthesized. Concentration-dependent [Ca2+]i responses were observed on human ASM cells stimulated with L1 (1 nM - 1 μM) that were comparable to the known contractile agonist histamine (10 μM). L1-induced [Ca2+]i response was significantly reduced in GPRC5B knockdown ASM cells. GPRC5B knockdown in ASM cells was verified by Western analysis. Further, L1 exposure increased the MLC phosphorylation (Thr18/Ser19) in human ASM cells compared to vehicle. Acute exposure of L1 (1 μM) increased airway contractility of PCLS (~17%-25%) compared to vehicle exposure. Conclusion: These findings suggest that GPRC5B activation modulates intracellular calcium and airway contractility in human ASM cells and murine PCLS. Thus, GPRC5B could potentially be considered for a novel therapeutic regimen to tackle asthma. This work was partly supported by NIH grants R01-HL146705 (Venkatachalem), R01-HL088029 (Prakash), and R01-HL142061 (Pabelick, Prakash). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cigarette smoking and vaping exacerbate asthma. Understanding the mechanisms of nicotine effects in asthmatic airways is important. This study demonstrates that functional alpha7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in human airway smooth muscle, including from asthmatics, and enhance intracellular calcium and contractility. Although a7nAChRs are associated with neuronal pathways, α7nAChR in smooth muscle suggests inhaled nicotine (e.g., vaping) can directly influence airway contractility. Targeting α7nAChR may represent a novel approach to alleviating airway hyperresponsiveness in asthma.