Thompson, M

Abstract Endothelial-to-mesenchymal transition (EndMT) is a biological process through which lung vascular endothelial cells (ECs) transdifferentiate into mesenchymal-like cells. EndMT has recently been implicated in the development and progression of pulmonary vascular remodeling in pulmonary hypertension (PH); however, its underlying regulatory mechanisms remain incompletely understood. MicroRNAs (miRNAs) are key post-transcriptional regulators of EC gene expression and cellular responses to various stimuli. Notably, microRNA-153 (miR-153) has been shown to directly target SNAI1 to modulate epithelial-to-mesenchymal transition (EMT), a process closely related to EndMT and extensively studied in cancer. Whether miR-153 also participates in EndMT regulation, however, remains unknown. In this study, we demonstrate that 72-hour hypoxic exposure induces SNAI1-mediated EndMT in human lung vascular ECs. Hypoxia also increased cell proliferation and disrupted intercellular junctions, leading to enhanced endothelial permeability. Reduced miR-153 expression was observed in both hypoxia- and TGF-β1-induced EndMT, as well as in ECs isolated from PH patients exhibiting an EndMT phenotype. Similar to hypoxia, TGF-β1 promoted EC permeability. Loss of miR-153 enhanced SNAI1-mediated EndMT, endothelial survival, and permeability under normoxic conditions, whereas miR-153 overexpression attenuated EndMT induced by hypoxia or TGF-β1. However, miR-153 restoration did not completely restore endothelial barrier integrity disrupted by these stimuli. In vitro findings were validated in experimental PH model. In conclusion, miR-153 serves as a critical regulator of EndMT, maintaining endothelial identity and barrier function. Therapeutic delivery of miR-153 may therefore represent a novel strategy to inhibit EndMT and attenuate pulmonary vascular remodeling in PH.

Abstract Rationale Premature birth necessitates interventions such as supplemental O2 (hyperoxia, typically <60% O2) with or without respiratory support primarily using nasal CPAP. The static stretch of CPAP can induce sustained airway hyperresponsiveness (AHR) and airway thickening (remodeling) even after CPAP is stopped - effects that contribute to a wheezing phenotype and predispose to asthma, major sources of respiratory morbidity in former “preemies.” Effects of CPAP can impact airway smooth muscle (ASM), a key cell type in asthma. We sought to find out if mechanosensitive Piezo (PZ) channels in developing ASM contribute to CPAP-induced AHR and remodeling. In other cell systems, PZ affects the Wnt/β-catenin (β-cat) pathway, but there is currently little information on how stretch influences this pathway and its downstream role in developing ASM. Our overall hypothesis is that in developing airways, PZ channels and downstream Wnt/β-cat pathway are linked in the context of stretch effects, leading to AHR and remodeling. Methods Fetal ASM (fASM) cells were isolated from lungs of 18-20-week fetuses (Mayo IRB exempt). Neonatal mice (comparable to late gestation humans) were also used; lung tissue was harvested following IACUC protocols. Western blot (WB), qRT-PCR, immunofluorescence imaging (IF), extracellular matrix (ECM) deposition assay, proliferation assay, and precision cut lung slice (PCLS) imaging were performed. Stretch conditions used were “non-stretch” (0% static strain imposed on 5% cyclic strain representing normal breathing) or “stretch” (5-10% static strain imposed on 5% cyclic strain, representing superimposed CPAP). Compounds used were Yoda1 (PZ1 agonist), GsMTx4 (PZ inhibitor), DKK1 (Wnt pathway inhibitor), and Wnt3a ligand. Results PZ1 and PZ2 are expressed in fASM and stretch increased expression of both, shown by qRT-PCR and WB. In neonatal mice exposed to CPAP, lumen area of PCLS were decreased compared to control; GsMTx4 alleviated this effect. Stretch increased ECM in fASM, however the effect was blunted by GsMTx4. Stretch increased mRNA for canonical Wnt pathway elements and decreased negative regulatory elements. Stretch increased GSK3β protein phosphorylation and β-cat protein nuclear translocation; effects blunted by DKK1. Wnt3a ligand increased proliferation and ECM in fASM. Stretch increased Wnt3a mRNA and β-cat protein nuclear translocation, with effects blunted by GsMTx4. Stretch alone or in combination with Wnt3a ligand, as well as Yoda1 alone, increased nuclear β-cat, shown qualitatively by IF. Conclusions Our study showed that Wnt/β-cat may be a novel, important, and conceivably targetable pathway (e.g. with PZ modulators) in alleviating potentially long-term, detrimental airway responses to CPAP. This abstract is funded by: NIH R01 HL056470 (Prakash), R01 HL177837 (Pabelick)

Abstract Rationale Asthma in the elderly (AIE) is characterized by hyperreactive and thickened, fibrotic airways (remodeling) that are not adequately addressed by current therapies. Thus, understanding mechanisms that drive AIE becomes 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)-Piezo1 and Piezo2 in the lung. While Pz channels are highly expressed, their specific contributions to ASM function remains largely unexplored. Moreover, Pz channels play essential roles in regulating the intracellular calcium homeostasis, which further drives mitochondrial dynamics. Whether Pz signaling influences mitochondrial structure and function-a key determinant of ASM energetics and contractility is unknown and therefore of significant interest. In the present study, we hypothesize that Pz1 channel activation regulates mitochondrial structure and function via an increase in intracellular calcium in ASM, leading to metabolic changes and remodeling, characteristic of AIE. Methods Primary human ASM cells were isolated from lungs of young (<45y) and AIE (≥65y) individuals obtained post-surgery (Mayo Clinic IRB approved). Cells were exposed to mechanical stretch (mimicking breathing) in the presence of either Yoda1/Jedi2 (agonist) or GsMTx4 (inhibitor) for 24 hrs. Mitochondrial fractionation was performed, and protein expression evaluated for mitochondrial dynamics targets (MFN1, MFN2, OPA1, DRP1 and FIS1) and transcriptional markers (mt-Cox1, mt-Cox2, ATP5b and TFAM). The colocalization of mitochondria and Pz channels was observed by immunofluorescence staining. Mitochondrial morphology was examined with MitoTracker staining while function was assessed using Seahorse XF analysis. Intracellular calcium ([Ca2+]i) and mitochondrial calcium ([Ca2+]m) were measured using 5µM Fura-2/AM and 1µM Rhod-2 AM respectively with fluorescence microscopy. Results Confocal microscopy revealed that Piezo1 localizes to mitochondria in ASM. Pz1 activation promoted mitochondrial fission, evidenced by an upregulation of DRP1/FIS1 and downregulation of MFN1/OPA1, confirming a fission-dominant phenotype. Pz1 activation also increased mitochondrial branching while reducing branch length, indicating enhanced fragmentation, particularly in AIE ASM. Yoda1 treatment reduced mitochondrial oxygen consumption rates, suggesting a lower energy demand. Furthermore, Yoda1 increased both [Ca2+]i and [Ca2+]m responses to histamine (10µM), with a more pronounced effect in AIE ASM. Conclusion Overall, our findings suggest that mechanotransduction through Pz channels, particularly Pz1 regulates intracellular calcium regulation and mitochondrial structure and function in ASM, linking extracellular mechanical forces to intracellular energy homeostasis. This mechanism may contribute to the altered energetics and remodeling observed in AIE airway pathophysiology. This abstract is funded by: 24POST1241979 (NAB), R01- HL056470 (YSP), R01-HL177837 (CMP)

Abstract Rationale Pulmonary Fibrosis (PF) is a fibroproliferative disease characterized by increased proliferation, constant extracellular matrix (ECM) deposition, and dysfunctional antioxidant systems. In PF, an emerging initiator is abnormal iron regulation and lipid peroxidation: consequences of iron catalyzed cell death, i.e. ferroptosis (FP). Abnormal iron regulation and lipid peroxidation lead to diminished glutathione (GSH)/glutathione peroxidase 4 (GPX4) antioxidant systems which promote ECM signaling in fibroblasts. Accordingly, inhibiting ferroptosis and/or correcting iron dysregulation via increasing GSH can help with early PF intervention. We hypothesize that enhanced hydrogen sulfide (H2S) signaling through exogenous H2S donors GYY4137 (slow-releasing) and AP39 (mitochondrial-specific), can blunt FP/lipid peroxidation leading to reduced ECM deposition in the context of PF. Methodsand Results Human fibroblasts were isolated from lung tissue of patients with PF and healthy patients (Mayo IRB approved) and were treated with exogenous H2S donors. Prussian blue staining of FFPE lung sections from patients with PF revealed increased ferric iron in alveolar regions. Immunostaining of PF human lung tissue showed lower expression of H2S generating enzyme, CBS, and higher expression of the lipid peroxidation enzyme, ACSL4, compared to non-PF patients. At the cellular level, Western blot was performed in fibroblasts to understand the effect of PF on Fe regulation. PF fibroblasts showed higher expression of transferrin receptor (TfR1) and ferritin, as well as higher expression of ACSL4. The impact of exogenous H2S donors on PF fibroblasts was tested. While H2S donors did not impact TfR1 or ferritin, they did blunt lipid peroxidation as determined using live cell BDP 581/591 C11- fluorescent imaging and ACSL4 protein expression. Furthermore, GYY4137 increased the cysteine transport protein xCT(SLC7A11; part of xC), the H2S synthesis enzyme CBS and GPX4, overall showing an antioxidant effect. This was further substantiated though measuring GSH/GSSG ratios via luminescence assay. In PF fibroblasts, GSH/GSSG levels were low comparatively to normal fibroblasts. However, with exogenous H2S, PF fibroblasts showed increased GSH/GSSG levels indicating a restoration of antioxidant function. Deposition of collagen-1 was higher in PF fibroblasts, and was blunted by GYY4137, which was more potent than the FP inhibitor ferrostatain-1. Conclusion Our data suggests ferroptosis/impaired antioxidant systems are significant factors in PF and that exogeneous H2S can be leveraged to replenish antioxidant systems and blunt FP, thus potentially reducing fibrotic activity. This abstract is funded by: NIH T32 HL 176414 (Ravi), R01 HL160570 (Pabelick, Prakash), R01 HL158532 (Prakash)