Pabelick, C.M.

Rationale: Pulmonary hypertension (PH) is a fatal disease characterized by a sustained elevated pulmonary vascular resistance that results in right heart failure. Despite therapeutic advances, no treatment provides disease progression reversion, resulting in poor long-term survival. Identifying new therapeutic targets for PH therefore requires a deeper understanding of its mechanisms. A defining feature of PH is pulmonary vascular remodeling, a result from excessive proliferation, migration, and apoptosis resistance of pulmonary artery smooth muscle cells (PASMC). SNAI1, a transcription factor regulating cell proliferation and differentiation during development and disease, has recently been implicated in endothelial dysfunction and endothelial-to-mesenchymal transition in PH. However, its role within PASMC remains unknown. Here, we aimed to define the contribution of SNAI1 in PASMC to the pathogenesis of PH. Methods: Normal human PASMC were exposed to normoxia (21% O 2 ) or hypoxia (3% O 2 ) for 72 hours. PASMC were also isolated from patients with or without PH. Cell proliferation and migration were assessed by EdU incorporation and wound scratch assays respectively. Mice were exposed to hypoxia (10% O 2 ) or Sugen5416/hypoxia (10% O 2 plus Sugen5416 20mg/kg, i.p., weekly) for 6 weeks. PH phenotype was characterized by right heart catheterization, right ventricle (RV) hypertrophy by Fulton index and pulmonary artery (PA) remodeling by H&E staining of lung tissues. Pharmacological blocker GN-25 was used to inhibit SNAI1 in vitro and in vivo. Results: SNAI1 expression was markedly increased in hypoxic PASMC and in PASMC from patients with PH. Hypoxia also upregulated HIF-1alpha, Vimentin, p-AKT, PCNA at the protein level in PASMC. EdU incorporation and migration rates were significantly higher in hypoxic cells as well as in PASMC isolated from PH patients compared with controls. In vitro, GN-25 (100 nM) abolished hypoxia-induced PASMC proliferation and migration. Right ventricular systolic pressure (RVSP), a surrogate measurement of PA systolic pressure, estimated mean PAP (mPAP), and RV contractility (RV-±dP/dtmax) were significantly increased in both PH models compared with controls. Additionally, Sugen5416/hypoxia-treated mice exhibited higher RVSP, mPAP and RV-±dP/dtmax compared to hypoxia-treated animals. The Fulton index (weight of RV vs. left ventricle and septum), was increased in both PH groups indicating development of RV hypertrophy in PH compared to control. PH mice lost body weight by 5-10%. Additionally, when Fulton index was normalized by body weight, RV hypertrophy was significantly higher in Sugen5416/hypoxia-treated mice compared with hypoxic animals. Lung tissues from PH mice exhibit PA hypertrophy (media remodeling) with some PA partially or completely occluded. Additionally, we detected protein upregulation of Snai1 and Vimentin in lung tissues of PH mice in both groups. Treatment of PH mice with GN-25 (20 mg/kg, daily, for the last 3 weeks under PH stimuli) in reversal experiments attenuated PH phenotype in both animal models by decreasing hemodynamic parameters, right ventricle hypertrophy and pulmonary vascular remodeling. Conclusions: Our findings identify a key role for SMC SNAI1 in the pathogenesis of lung vascular remodeling during hypoxia-induced PH. Inhibition of SNAI1 in PASMC may constitute a new therapeutic strategy for the treatment of PH. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Rationale: Respiratory disease is a major cause of mortality in Down syndrome (DS; trisomy 21, T21). DS is considered a progeroid syndrome, with elevated cellular senescence (SEN) and oxidative stress. Because SEN shapes organogenesis, its dysregulation may impair lung morphogenesis. We previously found SEN/SASP (senescence-associated secretory phenotype) abnormalities in prenatal human T21 lungs. Here, we test whether these alterations persist and change after birth. Methods: We analyzed human lung single-nuclear RNA (snRNA-seq) datasets (prenatal and postnatal) to quantify epithelial and mesenchymal SEN/SASP signatures. Sex- and age-matched T21 and non-T21 prenatal and postnatal lungs were studied; fibroblasts were isolated as prenatal human lung fibroblasts (HFLF) and postnatal human lung fibroblasts (HLF). SEN/SASP were measured by RT-qPCR and immunofluorescence (IF) in tissue and HFLF/HLF. HFLF/HLF were assayed for SA-β-gal, oxidative stress (CellROX/MitoSOX), and Ca 2 + dynamics (Fura-2 AM). 48h treatments with tunicamycin (1µM, SEN inducer), 4-phenylbutyrate (4-PBA, 2 mM, ER Stress inhibitor), or pyrroloquinoline quinone (PQQ, 20 μM, antioxidant) were performed on T21 and non-T21 HFLF/HLF. Conditioned media (CM) were collected after 48h from untreated cultures and then applied to cells of the opposite genotype for 24, 48, and 72h. Results: Overall SEN profile was similar in epithelium and mesenchyme across ages, but pathway signatures differed. Epithelium shows an inflammatory SASP, while mesenchyme shows a less-inflammatory, remodeling/growth-factor SASP. These lineage patterns were more evident postnatally. Whole-tissue IF showed increased γ-H2AX and p21 in T21 vs non-T21 both prenatally and postnatally (p< 0.05). At baseline, T21 HFLF/HLF had higher CDKN1A, CDKN2A, TP53, CDKN2B, and SERPINE1 (p< 0.05; n=5–6), with increased γ-H2AX/p21 IF (p< 0.01; n=6), SA-β-gal (p< 0.05; n=5–6), and ER/mitochondrial stress (p< 0.05; n=5–6). Fura-2 AM showed greater Ca 2 + release in T21 HFLF/HLF at baseline and after acetylcholine (10 μM, p< 0.01; n=5). Mitochondrial-morphology genes (FIS1, DNM1L, MFN1) were elevated in T21 HFLF/HLF (p< 0.05; n=6). In T21 HFLF, 4-PBA and PQQ each reduced SA-β-gal (p< 0.01; n=6). In postnatal HLF, PQQ had the stronger effect (p< 0.05; n=5). T21 CM induced progressively greater SEN/SASP over 24/48/72h in non-T21 cells (p< 0.05; n=5-6), while non-T21 CM partially rescued T21-HLF phenotypes at 24h only (p< 0.05; n=5-6). Conclusion: These data indicate that lung senescence in T21 initiates in utero and intensifies after birth and reflects lineage-specific pathways. ER-stress-related mechanisms are more prominent prenatally, while mitochondrial/redox mechanisms are predominant postnatally. This evidence suggests age and lineage-dependent drivers of senescence that may help explain DS lung disease and highlight potential new mechanistic targets. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Moderate hyperoxia (30%–60% O 2 ) in premature infants promotes bronchial airway hyperresponsiveness (AHR) via airway smooth muscle (ASM), a key regulator of bronchoconstriction, bronchodilation, and remodeling. Understanding how O 2 exposure drives long-term bronchial changes in prematurity is critical for developing therapies for airway disease across the lifespan. Premature lungs have immature antioxidant defenses, potentially due to disrupted mitochondrial dynamics, increasing susceptibility to O 2 -induced oxidative stress. Thus, mitochondrial homeostasis is highly relevant to ASM dysfunction and airway disease. We propose that hyperoxia in prematurity promotes mitochondrial dysfunction, and that the gasotransmitter hydrogen sulfide (H 2 S) mitigates O 2 -induced mitochondrial damage in developing ASM. Human fetal ASM (fASM) cells were exposed to moderate hyperoxia to investigate the effects of exogenous H 2 S donors (GYY4137, AP39) and stabilization of cystathionine β-synthase (CBS), an H 2 S biosynthetic enzyme, on mitochondrial structure and function. Hyperoxia impaired fASM cell mitochondrial integrity, while H 2 S donors in particular, or CBS stabilization attenuated adverse O 2 effects on mitochondrial morphology, ROS, respiration, calcium regulation, and contractility. These findings highlight the therapeutic potential of H 2 S in the premature lung exposed to moderate hyperoxia.

Abstract Rationale: While survival of premature infants has improved with NICU interventions, prematurity remains a risk for developing chronic lung diseases. Oxygen supplementation in the NICU is unfortunately necessary, and clinical practice has shifted towards use of moderate hyperoxia (&lt;60% O2) to minimize detrimental effects of very high O2 concentration (80-90%) such as bronchopulmonary dysplasia. However, even moderate hyperoxia increases the risk of bronchial airway diseases and wheezing/asthma with airway hyperreactivity and remodeling. We previously found that moderate hyperoxia induces cellular senescence in human fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms by which hyperoxia enhances cellular senescence in fASM are not known. We investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% (normoxia) vs. 50% O2 (hyperoxia). Methods: fASM isolated from lungs of 18-20 week aborted fetuses (Mayo IRB exempt; period of rapid bronchial airway growth) were exposed to normoxia, hyperoxia or etoposide (10 µM; positive control for senescence induction). Cells and lysates were harvested at days 2, 3 or 7 to assess markers of senescence markers, ER stress and mitochondrial morphology, and levels of cytosolic and mitochondrial ROS, using real-time fluorescence imaging, Western blotting, immunofluorescence, and a novel immuno-FISH staining of telomere associated foci (TAF). Mitochondria-specific antioxidant MitoQ [100 nM], ER stress inhibitor Salubrinal [12.5 µM] or mitochondrial fission inhibitor Mdivi-1 [10 µM] were used to assess their effects on senescence markers and interactions between ER stress and mitochondria in inducing senescence. Results: Hyperoxia enhanced cellular senescence via upregulation of p21, PAI-1, and Cav-1; downregulation of LMNB1 and Ki-67, with increased γH2A.X- and TAF-positive cells. Hyperoxia increased cytosolic and mitochondrial ROS, and markers of the three ER stress pathways (ATF6, IRE1α, XBP1s, PDI, GADD34). Hyperoxia altered mitochondrial morphology with increased fission protein Drp1 and fusion proteins Mfn1/2. Inhibition of Drp1 with Mdivi-1 blocked mitochondrial fragmentation, but not senescence. However, hyperoxia-induced p21 was reduced by the PERK pathway inhibitor Salubrinal and MitoQ. Conclusions: Hyperoxia induces senescence in fASM via mitochondrial ROS and ER stress: pathways that offer new opportunities to blunt detrimental effects of oxygen in developing lung via mitochondrial targeted antioxidants and/or ER stress inhibition.

Abstract Rationale: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and cellular chemical sensor. AhR maintains cellular homeostatsis in respons to endogenous and exogenous organic molecules. Airway smooth muscle (ASM) cells are major structural cells in airway which is involved in characteristic features of asthma such as increased airway wall thickness, airway remodeling and airway hyperresponsiveness. Previously, we found notable expression of AhR in human ASM and its increased expression during inflammation and asthma. However, the mechanisms of enhanced AhR expression in ASM is unknown. Therefore, we examined the role of pro-inflammatory signaling pathways in enhanced AhR expression. Methods: Primary human non-asthmatic and asthmatic ASM cells isolated from surgical lung resections (Mayo IRB approved) were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic. At ∼80% confluency, cells were serum deprived and treated with TNFα (20 ng/mL), IL13 (50 ng/mL) with or without PI3K/Akt inhibitor (Wortmannin: 50 nM), p38 inhibitor (SB203580: 600 nM), NFkB inhibitor (SN50 20 μM), and AP1 inhibitor (SR11302 10 μM). Subsequently, expression of AhR was determined using RNA sequencing, confocal microscopy, real-time qPCR, and Western blotting. AhR expression in isolated ASM fron lung tissue section was determined using laser capture microdissection (LCM). Moreover, role of AP1 in inducing AhR expression was confirmed using c-JUN (AP1) siRNA transfection in ASM cells. Additionally, binding of AP1 to the AhR promoter region was demonstrated by chromatin-immunoprecipitation (ChIP-qPCR) technique. Results: AhR expression was significantly high in asthmatic ASM cells and tissue than non-asthmatics. Exposure to pro-inflammatory cytokine TNFα increased AhR mRNA and protein expression, whereas IL-13 failed to do so. Furthermore, inhibition of p38 significantly reduced AhR expression whereas PI3K inhibition had no effect, indicating a role for p38 in AhR increased expression. AP1 inhibition significantly downregulated AhR, while NFkB inhibition showed no effect. Similarly, siRNA mediated silencing of c-JUN (AP1) also downregulated AhR, indicating AP1 role in AhR upregulation. Furthermore, transcription factor binding study suggested AhR promoter region has binding site for AP1 and NFkB. Interestingly, ChIP-qPCR study confirmed binding of AP1 at AhR promote region in response to TNFα. Conclusions: Collectively, the current study show that pro-inflammatory cytokine TNFα increases AhR expression through p38-AP1 pathway. The data shows that pro-inflammatory cytokines can regulate AhR expression in ASM, suggesting AhR in ASM has a role in asthma.

Abstract Rationale: Asthma in the elderly (AIE) presents a unique challenge characterized by hyperreactive and thickened, fibrotic airways (remodeling) that is not adequately addressed by current therapies. Thus, understanding mechanisms that drive AIE become important. Here, airway smooth muscle (ASM) cells play important roles in normal airway physiology and asthma pathophysiology. Recent discoveries have highlighted the significance of mechanosensitive Piezo (Pz) in health and disease. The lung generates mechanical forces of normal breathing and other maneuvers that are transmitted to bronchial airways via tethering parenchyma. While Pz channels are highly expressed in the lung, their specific role in ASM function remains largely unexplored. In this study, we used human ASM cells to test the hypothesis that increased airway stiffness associated with normal aging blunts Pz expression and mechanosensitive functionality, whereas asthmatic triggers (e.g. inflammation) enhance Pz expression and downstream signaling. Methods: Primary human ASM cells isolated from lungs of young (&lt;45y) vs. aged (&gt;65y) non-asthmatic vs. asthmatic individuals were obtained post-surgery (Mayo Clinic IRB approved). Cells were exposed to mechanical stretch (mimicking breathing) and mediators relevant to asthma (TNFα, IL-13) or fibrosis (TGFβ) to determine expression of Pz1/2 across the age spectrum. Separately, intracellular Ca2+ and contractility responses, proliferation and extracellular matrix (ECM) deposition across age/asthma groups were assessed in response to mechanical stretch and Pz1/2 modulation (inhibitors/activators, genetic approaches). Manipulation of Pz1/2 expression or function in ASM accompanied by mechanical stretch was used to assess impact on canonical Wnt/β-catenin signaling. Results: We observed increased Pz1/2 expression with age and asthma as well as with inflammation and mitogenic stimuli. Functionally, Pz1 agonist, Yoda1 induced increase in intracellular Ca2+, ASM proliferation and ECM deposition with greater effects observed in aging asthmatic ASM. Towards linking mechanosensation (via Pz) to remodeling via Wnt signaling, we find that Wnt3a is upregulated by Yoda1 but suppressed by Pz1 shRNA. Yoda1 stimulates TGFβ-induced Wnt3a and β-catenin, an effect further enhanced under mechanical stretch. Conclusions: By elucidating the role of Pz channels in ASM, we aimed to identify novel therapeutic targets for airway diseases. Understanding how mechanical forces and inflammatory stimuli influence Pz-mediated signaling pathways will lead to the development of innovative strategies to prevent or reverse airway remodeling in elderly asthmatic patients. Acknowledgement: Supported by 24POST1241979 (NAB) and NIH grants R01-HL142061 (CMP and YSP), R01- HL088029 (YSP).

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.