Christina M. Pabelick

Hypoxia is a well‐recognized risk factor in several pulmonary vascular diseases, including pulmonary hypertension (PH). Furthermore, hypoxia‐associated inflammatory changes enhance the structural and functional changes in the pulmonary artery (PA) of PH patients. Understanding the mechanisms that link hypoxia and inflammation, particularly early in disease, is key to the development of novel therapeutic avenues for PH. Thymic stromal lymphopoietin (TSLP) is an “early” inflammatory mediator thought to be critical in diseases such as asthma, chronic obstructive pulmonary disease, and atopic dermatitis. TSLP has canonical effects on the immune system, but can also have non‐canonical effects on resident lung cells, for example, airway smooth muscle. Currently, the expression and role of TSLP in the PA are unknown. We hypothesized that locally produced TSLP potentiates the effects of hypoxia in PA remodeling and contractility relevant to PH. Experiments in human PA endothelial cells (PAECs) and smooth muscle cells (PASMCs) found PAECs to be a larger source of TSLP, which targets PASMCs to enhance intracellular Ca ²⁺ responses and contractility to vasoconstrictor agonists, as well as cell proliferation and extracellular matrix deposition, acting via a number of signaling cascades including Stat3 and PI3/Akt. Hypoxia, acting via HIF1α, enhanced PAEC production of TSLP and promoted TSLP effects on PASMCs. Interestingly, TSLP per se enhanced HIF1α. Overall, these novel data highlight a role for TSLP in hypoxia effects on the PA, and thus relevance for inflammation in PH.

SiRNA delivery to target cellular processes is an appealing area in lung diseases such as asthma. To realize this potential requires efficient, targeted cytosolic delivery without cellular toxicity or siRNA degradation. We demonstrate the efficacy of PONI-Guan nanoparticles in accessing airway epithelial and smooth muscle cells with specific siRNA targeting of mRNAs of interest while maintaining barrier integrity and avoiding cellular toxicity.

Recognizing that antioxidant systems are impaired in the airways of premature infants, we used human fetal airway cells to explore the impact of oxygen on iron regulation and iron-mediated cell death (ferroptosis). We find moderate hyperoxia impairs antioxidant systems that prevent ferroptosis and dysregulates iron metabolism, while severe hyperoxia has a negative effect on mechanisms driving the early onset of ferroptosis. Inhibitors of ferroptosis decrease iron and lipid peroxidation, demonstrating links between oxygen and iron regulation in developing airways.

Iron is essential for cellular function, and lung cells are no exception. Previous studies have demonstrated an association between increased level of iron in the lung and aging and age-related lung diseases including pulmonary fibrosis and asthma in the elderly (AIE). However, the mechanisms underlying the accumulation of iron with aging or AIE, or the cell types involved, remain understudied. In the context of asthma, airway smooth muscle is a key cell type contributing to contractility as well as airway remodeling (proliferation, fibrosis). In this study we characterized iron level and regulation in human (hASM) from young (2+ ) were determined using the fluorescent dye FerroOrange. Cell lysates were analyzed for iron accumulation, antioxidant, lipid peroxidation, and extracellular matrix (ECM) markers, and cell proliferation was assessed. We found that iron accumulates with aging, but surprisingly decreases with AIE. hASM from AIE patients showed activated antioxidant pathways and lipid peroxidation, while FAC-exposure impaired iron metabolism and enhanced ECM deposition. Iron depletion mitigated the antioxidant response, lipid peroxidation, cell proliferation, ECM production. These findings suggest that iron metabolism in hASM contributes to cell hyperplasia and ECM, while in aging and particularly AIE, counter-regulatory changes in iron metabolism and antioxidant pathways occur, overall promoting airway remodeling.

Iron is an important metal element regulating biological processes such as gene expression, electron transport, cell proliferation, differentiation and division, and cellular respiration. Regulation of iron levels is therefore critical, with excess iron accumulation leading to iron-dependent lipid peroxidation and cell death (ferroptosis). Factors such as aging and environmental exposures (e.g. high/low oxygen, particulate iron) can disrupt iron homeostasis. Iron-dependent processes influence downstream pathways such as inflammation, mitochondrial energetics, and remodeling (proliferation, fibrosis): pathways involved in lung diseases particularly at the extremes of age: from hyperoxic lung injury in neonates to chronic diseases such as asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis (PF) that increase with age. While these diseases involve structural and functional changes in bronchial vs. alveolar compartments, including airway hyperreactivity, altered mucus production, cell proliferation and migration, increased extracellular matrix (ECM) remodeling, senescence and metabolic dysregulation, the casual or consequential links between iron dysregulation and disease pathology, particularly with aging, are still being investigated. The aim of this topical review is to (i) highlight current knowledge regarding iron regulation in normal lung vs. lung diseases, (ii) identify known roles of iron in lung homeostasis, and the contribution of iron dysregulation in lung diseases with aging, and (iii) explore the potential benefit of targeting iron regulation and/or ferroptosis in lung diseases.

As an intrinsically mechanosensitive organ, the lung experiences a range of mechanical forces. Chronic obstructive pulmonary disease (COPD) involves abnormal macroscopic cellular and extracellular matrix (ECM) changes that impact mechanical properties of the lung. Mechanosensitive Piezo1/2 channels are expressed in the lung including on airway smooth muscle cells (ASM) that mediate cellular responses to stretch and ECM biomechanics. The expression and roles of Piezos in COPD lung ASM are not known. We hypothesized that Piezo expression and activation are altered in COPD lung ASM influencing ECM regulation. Distribution of Piezo proteins in ASM and epithelium of small airways of COPD stage II and IV vs. non-COPD controls was assessed using immunohistochemistry and ImageJ (n=10-17/group). Isolated ASM cells from control (n=6) vs. COPD stage II and IV patients (n=3 each stage) were exposed to stretch or the Piezo1 agonist Yoda1 followed by measurement of ECM gene and protein expression. Less Piezo2 staining was observed in COPD IV patients compared to controls, with lesser area and intensity of staining in the epithelial layer, and lower intensity of staining in ASM and small airways as a whole. Fura-2-based imaging of ASM Ca ²⁺ showed lower influx after Yoda1 exposure in COPD II compared to control and COPD IV. Gene expression of Piezo1 increased upon stretching in controls but not in COPD ASM, while Piezo2 protein expression decreased with stretching in all groups. Yoda1 treatment resulted in decreased collagen1 , fibulin1 and periostin gene and collagen 1 and periostin protein expression in ASM. Overall, these results support a role for Piezo activation in abnormal ECM-ASM cell crosstalk in COPD.

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.

The studies demonstrate the differential effects of the nicotinic acetylcholine receptors (nAChRs) regulating airway hyperreactivity (AHR) and remodeling in a mixed allergen model of asthma. We compared wild-type (WT) versus global α7nAChR knockout (Glα7KO) versus smooth muscle-specific α7nAChR KO (smα7KO) mice. We found that smα7KO improved lung function and AHR in mice challenged with MA and in the presence of nicotine, whereas MA-challenged Glα7KO in the presence of nicotine showed worsened AHR.

Mechanical forces associated with positive-pressure respiratory support used in the intensive care of preterm infants can have both positive and inadvertent consequences for lung development. Positive pressure support improves pulmonary gas exchange, limits atelectasis, and improves systemic oxygenation, but the injurious effects of invasive modalities, such as mechanical ventilation, on lung development are also well known. However, there are major gaps in our understanding of the adverse effects of less invasive forms of ventilation. Here, continuous positive airway pressure (CPAP) is one of the most common respiratory support modalities, representing the least invasive of the positive pressure spectrum. A major limitation in studying human airway development is the lack of age-appropriate models and the ability to administer CPAP to small neonatal animals. We resolved this problem by developing a mouse model of neonatal CPAP delivered daily to awake, un-anesthetized newborn mice -- a model that utilizes clinically relevant CPAP levels at a stage of mouse lung development that corresponds with preterm infants when they are likely to receive CPAP in the ICU. Here, we demonstrate our CPAP model, which includes a convenient, custom-designed system that can deliver CPAP non-invasively to the un-anesthetized newborn with easily adjustable levels. We also provide a demonstration of the precision cut lung slice method for the measurement of airway hyperreactivity ex vivo. The PCLS is described here because it provides a convenient functional readout of the pulmonary system, which can be technically challenging in small (neonatal) animal models, and also has wide applicability to other models unrelated to CPAP. We show data demonstrating that CPAP elicits airway hyperreactivity, which may have important clinical implications for the choice of respiratory support modality in the intensive care of preterm infants.

Mechanical forces associated with positive-pressure respiratory support used in the intensive care of preterm infants can have both positive and inadvertent consequences for lung development. Positive pressure support improves pulmonary gas exchange, limits atelectasis, and improves systemic oxygenation, but the injurious effects of invasive modalities, such as mechanical ventilation, on lung development are also well known. However, there are major gaps in our understanding of the adverse effects of less invasive forms of ventilation. Here, continuous positive airway pressure (CPAP) is one of the most common respiratory support modalities, representing the least invasive of the positive pressure spectrum. A major limitation in studying human airway development is the lack of age-appropriate models and the ability to administer CPAP to small neonatal animals. We resolved this problem by developing a mouse model of neonatal CPAP delivered daily to awake, un-anesthetized newborn mice -- a model that utilizes clinically relevant CPAP levels at a stage of mouse lung development that corresponds with preterm infants when they are likely to receive CPAP in the ICU. Here, we demonstrate our CPAP model, which includes a convenient, custom-designed system that can deliver CPAP non-invasively to the un-anesthetized newborn with easily adjustable levels. We also provide a demonstration of the precision cut lung slice method for the measurement of airway hyperreactivity ex vivo. The PCLS is described here because it provides a convenient functional readout of the pulmonary system, which can be technically challenging in small (neonatal) animal models, and also has wide applicability to other models unrelated to CPAP. We show data demonstrating that CPAP elicits airway hyperreactivity, which may have important clinical implications for the choice of respiratory support modality in the intensive care of preterm infants.