Christopher D. Pascoe

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is a genetic disorder characterized by progressive neuromuscular and multisystem symptoms. MELAS typically manifests during childhood, can be difficult to diagnose, and has no cure. Extracellular vesicles (EVs) are lipid-enclosed nanoparticles secreted from cells that contain biological cargo and have demonstrated potential as biomarkers. We investigated the potential of plasma-derived EVs as diagnostic biomarkers of MELAS and examined their functional effects on mitochondrial respiration in treated skeletal muscle myotubes. Plasma-derived EVs were isolated from MELAS patients and age- and sex-matched control individuals. The biophysical characteristics and cargo of the EVs were analyzed. A Mito Stress Test was performed to assess oxygen consumption rate (OCR) in healthy myotubes treated with Control- or MELAS-EVs. Nine MELAS patients from two families were studied, and the results were categorized by age, sex and heteroplasmy level. EV size and zeta potential remained unchanged. However, total EV concentration was higher in MELAS patients, particularly for small-EVs (<200 nm) and in younger patients (<25 years old). Relative protein yield per EV was lower in the MELAS group, especially among female and younger individuals. EV double-stranded DNA (dsDNA) concentration did not differ between MELAS- and Control-EVs overall, but was higher in male MELAS patients. Protein markers typically enriched in small EVs showed altered expression in MELAS EVs: TSG101 and CD63 were lower, while flotillin-1 was higher compared to Control-EVs. A decrease in basal OCR was shown in cells treated with MELAS-EVs, with a similar response noted in the group treated with EVs from female MELAS patients. Post-treatment analysis showed no differences in oxidative phosphorylation (OXPHOS) subunit levels between cells treated with MELAS- and Control-EVs. In conclusion, plasma-derived EVs show promise as potential biomarkers for MELAS, and circulating EVs in this patient population may contribute to systemic metabolic dysfunction.

One-lung ventilation (OLV) is performed during lung surgeries by ventilating a single lung, while collapsing the operative lung to provide surgical exposure within the thoracic cavity. While a lung-protective ventilation strategy is recommended during OLV, increasing the fraction of inspired oxygen (FiO₂) or tidal volume (V_T) may be required to prevent hypoxemia during surgery. Unfortunately, these increases are associated with postoperative lung injury. Using a porcine model of OLV, our project aims to determine if high FiO₂ or V_T during OLV contributes to elevation of pro-inflammatory lipid mediators postoperatively. Fifteen three-month-old farm-bred pigs underwent left upper lobectomy requiring OLV. Pigs were exposed to one of three ventilation parameters: normoxic low V_T lung-protective ventilation LPV-NO, n = 5, FiO₂ < 50%, V_T = 6 mL/kg), hyperoxic lung-protective ventilation (LPV-HO, n = 5, FiO₂ >100%, V_T = 6 mL/kg), or normoxic high V_T (injurious mechanical ventilation) (IMV, n = 5, FiO₂ < 50%, V_T = 10–12 mL/kg). Arterial plasma was collected before and after OLV, and lipids were detected via LC-MS-MS. Lipidomic analysis demonstrated a statistically significant increase (FC = 2, p ≤ 0.05) in lysophosphatidylethanolamines (LPE 18:3, LPE 20:4, LPE 18:2, LPE 22:6), free fatty acids (FFA 20:4), phosphatidylserine (PS 38:5), lysophosphatidylcholine (LPC 18:1, LPC 18:3, LPC 22:6), triglyceride (TG 18:2-18:2-20:4), free fatty acids (FFA 20:5), linoleyl-carnitine molecules (C18-2 Linoleoyl Carnitine), and phosphatidylethanolamines (PE 36:5) in LPV-HO. IMV resulted in a significant increase (FC = 2, p ≤ 0.05) in triglyceride (TG 18:2-18:2-20:4), diglyceride (DG 18:1-20:4, DG 16:0-20:4), linoleyl-carnitine molecules (C18-2 Linoleoyl Carnitine), and free fatty acids (FFA 20:5). There was no significant change in lipid biomarker levels following LPV-NO post-surgery. Our lipidomic analysis supports that both high FiO₂ and V_T contribute to systemic lipid metabolic derangements. Lipids that were elevated in LPV-HO and IMV are associated with multiple inflammatory pathways implicated in lung injury. This suggests that intra-operative anti-inflammatory therapies targeted to these lipid pathways may reduce or prevent postoperative pulmonary complications after lung surgery.

Chronic obstructive pulmonary disease (COPD), the fourth leading cause of death worldwide, is traditionally considered a disease of smoking. However, <20% of people who smoke develop COPD, indicating the disease is complex, resulting from the interplay of genetic and environmental factors. Emerging evidence highlights the importance of exposure in early life to environmental irritants that impair fetal lung development and subsequent lung function trajectories, increasing risk for future COPD. Specifically, childhood asthma, preterm birth, and surfactant deficiency have been associated with lung function impairments and an increased COPD risk later in life. Furthermore, prenatal exposure to cigarettes influences sensitivity of individuals to smoking in their later life. A mounting body of evidence now indicates that diabetes exposure during pregnancy increases the risk for several childhood conditions linked to COPD risk, suggesting that maternal diabetes may be an unexplored risk factor for COPD. This article reviews the current literature on the influence of maternal diabetes on known early-life COPD risk factors (asthma and preterm birth), and identifies knowledge gaps that need to be addressed to pindown a potential association with COPD. Specifically, whether exposure to maternal diabetes influences offspring risk for COPD through already identified risk modifiers, or directly by altering lung function trajectories or sensitivity to cigarettes. Maternal diabetes rates are rising worldwide, with type 2 diabetes mellitus (T2DM) during pregnancy and gestational diabetes mellitus (GDM) nearly doubling over the last 15 years. Understanding how prenatal diabetes influences COPD risk is imperative to establishing whether intervening early can prevent COPD in this population.

Oxylipins, diverse lipid mediators derived from fatty acids, play key roles in respiratory physiology, but the contribution of lung structural cells to this diverse profile is not well understood. This study aimed to characterize the oxylipin profiles of airway smooth muscle (ASM), lung fibroblasts (HLF), and epithelial (HBE) cells and define how they shift when they are exposed to stimuli related to contractility, fibrosis, and inflammation. Using HPLC-MS/MS, 162 oxylipins were measured in baseline media from cultured human ASM, HLF, and HBE cells as well as after stimulation with modulators of contractility and central regulators of fibrosis/inflammation. At the baseline, ASM and HLF cells had the most similar oxylipin profiles, dominated by oxylipins from cytochrome P450 (CYP450) epoxygenase metabolites. TGFβ stimulation of HLF suppressed CYP450-derived oxylipins, while ASM stimulation increased prostaglandin production. HBE showed the most distinct baseline profile enriched with cyclooxygenase (COX)-derived oxylipins. TGFβ stimulation of HBE increased the level of several oxylipins from CYP450 epoxygenases. These findings highlight the importance of CYP450 oxylipins, which are relatively unexplored in the context of respiratory physiology. By resolving these oxylipin profiles, we enable future respiratory research to understand the function of these oxylipins in regulating physiology, especially in the context of modifying contraction and inflammation.

Oxidative stress is associated with asthma pathobiology. We reported that oxidized phosphatidylcholines (OxPCs) are mediators of oxidative stress and accumulate in the lung in response to allergen challenge. The current study begins to unravel mechanisms for OxPC accumulation in the lung, providing the first insights about how OxPCs underpin allergic airway pathophysiology, and pre-clinical testing of selective neutralization of OxPCs in a murine model of allergic asthma. We hypothesized that intranasal delivery of E06, a natural IgM antibody that neutralizes the biological activity of OxPCs, can ameliorate allergen-induced airway inflammation and airway hyperresponsiveness. Adult BALB/c mice were intranasally (i.n.) challenged with house dust mite (HDM) (25 μg/mouse, 2 weeks). Some animals also received E06 monoclonal antibody (mAb) (10 µg) i.n. 1 hr before each HDM challenge. HDM challenge reduced mRNA for anti-oxidant genes (SOD1, SOD2, HO-1, and NFE2L2) in the lung by several orders of magnitude (p < 0.05). Concomitantly, total immune cell number in bronchoalveolar lavage fluid (BALF) increased significantly (p < 0.001). E06 mAb treatment prevented allergen-induced BALF immune cell number by 43% (p < 0.01). This included a significant blockade of eosinophils (by 48%, p < 0.001), neutrophils (by 80%, p < 0.001), macrophages (by 80%, p < 0.05), and CD4 (by 30%, p < 0.05) and CD8 (by 42%, p < 0.01) lymphocytes. E06 effects correlated with a significant reduction in TNF (by 64%, p < 0.001) and IL-1β (by 75%, p < 0.05) and a trend to diminish accumulation of other cytokines (e.g., IL-4, -10, and -33, and IFN-γ). E06 mAb treatment also inhibited HDM exposure-induced increases in total respiratory resistance and small airway resistance by 24% and 26%, respectively. In conclusion, prophylactic treatment with an OxPC-neutralizing antibody significantly limits allergen-induced airway inflammation and airway hyperresponsiveness, suggesting that OxPCs are important mediators of oxidative stress-associated allergic lung pathophysiology.

Smoking is a potent cause of asthma exacerbations, chronic obstructive pulmonary disease (COPD) and many other health defects, and changes in DNA methylation (DNAm) have been identified as a potential link between smoking and these health outcomes. However, most studies of smoking and DNAm have been done using blood and other easily accessible tissues in humans, while evidence from more directly affected tissues such as the lungs is lacking. Here, we identified DNAm patterns in the lungs that are altered by smoking. We used an established mouse model to measure the effects of chronic smoke exposure first on lung phenotype immediately after smoking and then after a period of smoking cessation. Next, we determined whether our mouse model recapitulates previous DNAm patterns observed in smoking humans, specifically measuring DNAm at a candidate gene responsive to cigarette smoke, Cyp1a1. Finally, we carried out epigenome-wide DNAm analyses using the newly released Illumina mouse methylation microarrays. Our results recapitulate some of the phenotypes and DNAm patterns observed in human studies but reveal 32 differentially methylated genes specific to the lungs which have not been previously associated with smoking. The affected genes are associated with nicotine dependency, tumorigenesis and metastasis, immune cell dysfunction, lung function decline, and COPD. This research emphasizes the need to study CS-mediated DNAm signatures in directly affected tissues like the lungs, to fully understand mechanisms underlying CS-mediated health outcomes.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2–loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Prenatal and early-life exposure to cigarette smoke (CS) has repeatedly been shown to induce stable, long-term changes in DNA methylation (DNAm) in offspring. It has been hypothesized that these changes might be functionally related to the known outcomes of prenatal and early-life CS exposure, which include impaired lung development, altered lung function, and increased risk of asthma and wheeze. However, to date, few studies have examined DNAm changes induced by prenatal CS in tissues of the lung, and even fewer have attempted to examine the specific influences of prenatal versus early postnatal exposures. Here, we have established a mouse model of CS exposure which isolates the effects of prenatal and early postnatal CS exposures in early life. We have used this model to measure the effects of prenatal and/or postnatal CS exposures on lung function and immune cell infiltration as well as DNAm and expression of Cyp1a1, a candidate gene previously observed to demonstrate DNAm differences on CS exposure in humans. Our study revealed that exposure to CS prenatally and in the early postnatal period causes long-lasting differences in offspring lung function, gene expression, and lung Cyp1a1 DNAm, which wane over time but are reestablished on reexposure to CS in adulthood. This study creates a testable mouse model that can be used to investigate the effects of prenatal and early postnatal CS exposures and will contribute to the design of intervention strategies to mediate these detrimental effects. NEW & NOTEWORTHY Here, we isolated effects of prenatal from early postnatal cigarette smoke and showed that exposure to cigarette smoke early in life causes changes in offspring DNA methylation at Cyp1a1 that last through early adulthood but not into late adulthood. We also showed that smoking in adulthood reestablished these DNA methylation patterns at Cyp1a1, suggesting that a mechanism other than DNA methylation results in long-term memory associated with early-life cigarette smoke exposures at this gene.