Background:During the development of idiopathic pulmonary fibrosis (IPF), elastin is broken down and replaced with a stiffer substrate which interferes with normal breathing. This remodeling releases elastin degradation products (EDPs), or elastokines, which can then be measured. We examined the association between elastokine concentrations and disease severity to determine if EDP concentrations are associated with clinically relevant markers in IPF. Methods:Concentrations of elastokines were measured via enzyme linked immunosorbent assay (ELISA). Samples were obtained from a single institution's Interstitial Lung Disease (ILD) registry/biorepository (n = 81 with IPF and 24 healthy volunteers). We used linear and logistic regression modeling to assess the association between EDP concentrations at the time of diagnosis, lung function, and clinical outcomes. Results:Patients with IPF were older, more likely to be male, had ever smoked, and had worse lung function compared to healthy volunteers (p value ≤ 0.02 for all parameters). Patients with IPF had higher concentrations of elasotkines (p < 0.001), and the highest elastokine concentrations were associated with reduced forced vital capacity (FVC, p = 0.026), and decreased three-year transplant-free survival (p = 0.0005). These findings suggest that elastokines are biomarkers of matrix turnover and are associated with relevant clinical outcomes in patients with IPF.

Abstract Background Metabolic syndrome (MetS) is highly prevalent worldwide, affecting up to one-third of adults. Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease, with a global prevalence of 17.7 per 100,000 persons. Given the ubiquity of MetS in general population, its coexistence with IPF is likely, and may influence disease outcomes. This study examined whether having both IPF and MetS worsens respiratory and metabolic outcomes compared to IPF without MetS, using real-world data. Methods The TriNetX Global Collaborative Network was queried to identify patients with IPF diagnosed between 1/2020-1/2025. MetS was defined by ≥ 3: type 2 diabetes mellitus, hypertension, dyslipidemia and BMI ≥ 30 kg/m2. Only 1.78% of participants had a recorded standard diagnostic code for MetS on TriNetX; therefore, proxy criteria were applied to identify additional cases. Comparison groups included: IPF with MetS vs IPF without MetS. Propensity score matching (PSM, 1:1) was used to make matched groups for age, gender, race, tobacco/alcohol use, emphysema, chronic obstructive pulmonary disease, chronic kidney disease. Key outcomes included major adverse cardiovascular events (MACE), major adverse liver outcomes (MALO), respiratory outcomes and all-cause mortality, and these were tracked up to 5 years. Results 21,800 patients diagnosed with IPF and MetS, and 20,834 patients with IPF without MetS were identified. After PSM, there were 17,018 patients in each group. Patients with IPF and MetS had a higher odds of acute respiratory failure (OR 1.5), chronic respiratory failure (OR 1.47), pulmonary hypertension (OR 1.41) and pulmonary embolism (OR 1.3). MACE risk was elevated in those with IPF and MetS group (OR 1.77). The risk of MALO, specifically ascites and encephalopathy were higher in patients with IPF with MetS (OR 1.45). Hospitalizations (OR 1.57) and all-cause mortality (OR 1.04) were increased in patients with IPF and MetS. Conclusions Presence of MetS in patients with IPF was associated with higher odds of adverse pulmonary, cardiovascular, liver-related outcomes, as well as increased mortality. These findings highlight the need for comprehensive cardiometabolic risk assessment and management in patients with IPF. This abstract is funded by: None

Abstract Background Radiation-induced lung injury (RILI) occurs in cancer patients treated with thoracic radiation. Although 5-10% of cancer survivors develop clinically significant symptoms like dry cough or dyspnea, up to 90% of patients develop signs of RILI on CT. These fibrotic lesions can obscure tumor recurrence or impact treatment for recurrence. Our lab was the first to link RILI to metabolic dysfunction using metabolomic analysis of exhaled breath condensate, including elevated lactate production, which drives fibroblast-to-myofibroblast transdifferentiation (FMT), a key process in fibrosis. Here, we investigate whether ionizing radiation drives additional metabolic reprogramming in lung fibroblasts and if these processes can be therapeutically targeted. Methods Primary human lung fibroblasts (HLFs) were irradiated with 2-5 Gy ionizing radiation from an X-ray source to induce FMT and were harvested after 5 days. Protein expression was determined by western blot, and lactate was measured in conditioned medium by a colorimetric assay. Glycolysis rates were measured using the Seahorse Xfe system. 13C-labelled glucose or ribose was added to medium as the only sugar for 4 hours prior to harvest, and targeted metabolomics analysis was performed on cell lysates by liquid chromatography/tandem mass spectrometry. To investigate potential therapeutic targets, irradiated HLFs were treated with shikonin to inhibit pyruvate kinase M2 (PKM2), or with N3-pyridyl thiamine (N3PT) to inhibit transketolase (TKTL), and FMT was assessed by immunocytochemistry. Results Radiation markedly induces alpha-smooth muscle actin and extracellular lactate (Fig. 1A- B). Radiation significantly accelerates both basal and compensatory glycolysis (Fig. 1C). Radiation decreases glucose-derived glycolysis products but increases ribose-derived glycolysis products, as shown by analysis of labeled phosphoenolpyruvate (Fig. 1D- E). Ribose is a significant source of lactate in irradiated fibroblasts (Fig. 1F). Inhibition of PKM2, the final step in glycolysis, attenuates FMT and lactate accumulation (Fig. 1G-H and data not shown). Surprisingly, inhibition of TKTL, which catalyzes the entry of ribose products into glycolysis, also inhibits FMT and lactate secretion (Fig. 1G-H and data not shown). Conclusions We observed that radiation-induced FMT of primary HLFs is marked by glycolytic reprogramming with increased rates of glycolysis and lactate production that is fueled, in part, by the pentose phosphate pathway. This supports results from RILI patients, suggesting that reverse flux through the pentose phosphate pathway compensates for the increased energy demand of FMT and the diversion of glycolysis products to lactate. Pharmacologically targeting these pathways may be a useful therapeutic approach to treat fibrosis by depriving fibroblasts of fuel. This abstract is funded by: This work was supported in part by National Institute of Health (NIH) Grant R01HL127001. J.P.-L.O. was funded in part by a Ford Foundation Predoctoral Fellowship. Services in support of the research project were provided by the VCU Massey Cancer Center Lipidomics and Metabolomics Shared Resource, which is supported, in part, with funding from NIH-National Cancer Institute Cancer Center Support Grant P30 CA016059. P.D.J. was supported by the Pulmonary Fibrosis Foundation Scholars program. M.A.T.F was supported in part by the Pulmonary Fibrosis Foundation Scholars Program and by NIH grant K99HL169903. This content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Abstract Rationale Idiopathic Pulmonary Fibrosis (IPF) is an insidious, progressive restrictive lung disease contributing to significant loss of life and quality of life for approximately 5 million individuals globally with poorly understood etiology, few therapies, and no known cure. One of the hallmarks of IPF is a phenotypic change of fibroblasts into myofibroblasts, which contribute to differential extracellular matrix (ECM) production, leading to the scarring and stiffening of the lung that drives the progressive pulmonary dysfunction and death. Our lab has previously demonstrated that an increase in tissue stiffness leads to the progressive development of a fibrotic phenotype in fibroblasts, but the underlying intracellular mechanotransduction response to changing tissue stiffness remains uncharacterized. This study aimed to clarify the intracellular signaling cascade in response to stiffness for potential development of future antifibrotic therapies. Methods Primary Human Lung Fibroblasts (HLFs) were cultured on collagen coated substrate with differing stiffnesses (1 kPA, 60 kPA, and tissue culture plastic). After five days, whole cell protein lysate was placed on a Phospho Explorer Antibody Microarray (Full Moon Biosystems) following the manufacturer’s protocol. Fold change protein phosphorylation was calculated and Pathway Enrichment Analysis was conducted in String. Results Pathway enrichment analysis in STRING highlighted significant interactions among 51 proteins influenced by stiffness. The analysis isolated clusters related to cell proliferation, survival, repair, and cell cycle gatekeeping (Fig 1A). HLF cells grown on softer substrates relative to plastic exhibited notable posttranslational modification of proteins involved in DNA regulation and repair with an increase in phosphorylation of p53, Calmodulin, CREB, JUN, CHK2, and SMC1 and dephosphorylation of BRCA1, CDC25A, and CaMK4 (Fig 1B). Furthermore, proliferation, adhesion and migration signals including EGFR, ICAM1, SRC, MEK, and PXN were increasingly phosphorylated while RAF1, MKK7 and PYK2 were dephosphorylated on softer substrates (Fig 1C). Conclusions These findings suggest that tissue stiffness affects cellular survival and proliferation via the DNA damage repair and cell cycle regulation pathways, calcium signaling via calmodulin, and proliferation, adhesion, and migration pathways. These data help define the intracellular response to phenotypic changes previously described in response to substrate stiffness in fibrosis and implicate a complex interplay between tyrosine kinase receptors and tissue stiffness in fibrotic progression. Modulation of cellular mechanosensation may offer a new direction for antifibrotic therapies. In the future, we plan to investigate whether targeting these pathways may provide synergistic benefit with current tyrosine kinase inhibitor therapeutics. This abstract is funded by: This work was supported in part by the NIH, the Pulmonary Fibrosis Foundation, and the Parker B. Francis foundation. MATF is funded by Parker B. Francis Foundation and NIH grant K99HL169903-01A10. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Background: Idiopathic Pulmonary Fibrosis (IPF) is a progressive scarring disease of the lung driven by multiple feed-forward mechanisms. Tissue stiffening and metabolism have independently been identified as important pathogenic drivers. Fibroblasts sense increased stiffness present in fibrotic tissue and respond with pro-fibrotic phenotypic changes. Piezo2 is one possible mechanosensor fibroblasts use to sense the stiffness of their environment. Here we investigate the role of Piezo2 activity on intracellular metabolism. We hypothesize that Piezo2 activity regulates cellular metabolic reprogramming of stiffness-induced myofibroblast differentiation. Methods: Primary HLFs from non-fibrotic donors were seeded on collagen-coated substrates of 2-kPa (healthy) and 64-kPa (fibrotic) stiffness (n = 3/treatment). Cells were treated with the Piezo2 inhibitor D-GsMTx4 (10 µM). Cell lysates were collected for untargeted metabolomics analysis. Untargeted metabolomic analysis identified 716 unique compounds, 178 of which were matched to named metabolites. The metabolite list was further filtered to 102 metabolites based on statistically significant (p<0.05) changes due to Piezo2 activity (D-GsMTx4 treatment) or due to changes in substrate stiffness. Results: We observe significant shifts in metabolism due to both Piezo2 activity and mechanical environment (Figure 1). Partial Least Squares analysis demonstrates tight clustering of the treatment groups (Fig. 1A). Custer 1 highlights metabolites which increase in response to stiffness and decrease to baseline with piezo2 inhibition, while cluster 2 highlights metabolites that decrease with increasing stiffness, but return to baseline with piezo2 inhibition (Fig. 1B). These metabolites represent the following processes related to energy metabolism and protein synthesis, converging on glutamate (Fig. 1C,D). Clusters 3 and 4 represent metabolites that increase or decrease with DGS treatment regardless of stiffness. These metabolites are implicated in oxidative stress response. Discussion: Fibroblasts are primary effector cells in pulmonary fibrosis. Here we demonstrate that inhibiting Piezo2 activity alters cell metabolism, identifying glutamate metabolism as a key pathway. Our results show that inhibition of piezo2 reverses probiotic changes in HLFs by making them unable to sense the increasing stiffness of their environment. The metabolic changes identified relate to collagen synthesis and energy metabolism to which potentially feed the demands of fibrotic proliferation, differentiation, migration, and matrix production. Leveraging the intersection between altered cellular metabolism and mechanotransduction mechanisms can be critical to identifying novel therapeutic targets to treat pulmonary fibrosis. We conclude that HLFs use Piezo2 to sense their environment, leading to pro-fibrotic changes and blocking Piezo2 may be a viable therapeutic option.

Rationale: The term fibrotic interstitial lung disease (f-ILD) is an umbrella term that includes several conditions such as idiopathic pulmonary fibrosis (IPF), fibrotic hypersensitivity pneumonitis (f-HP) and systemic autoimmune disease such as Systemic Sclerosis – associated ILD (SSc-ILD) that have significant morbidity and mortality. Distinguishing between these conditions has significant therapeutic implications but it can be challenging given similarities in presentation. Bioactive lipids such as sphingolipids are crucial structural elements of cellular membranes and participate in signal transduction; they have recently been implicated in the pathogenesis of f-ILD. The aim of this study was to identify a plasma sphingolipid profile which would aid in differentiation of these disease states. Methods: Using Virginia Commonwealth University's ILD biorepository we identified individuals with IPF, f-HP, and SSc-ILD and healthy controls. Each patient's diagnosis was verified by multidisciplinary ILD conference. Platelet poor plasma was analyzed for sphingolipid analysis using high performance liquid chromatography tandem mass spectrometry. All specimens were obtained prior to initiation of any anti-inflammatory or anti-fibrotic therapy. Patient's demographic and radiographic data were extracted from the biorepository database. Results: Data from 60 patients, (20=f-HP, 10=SSc-ILD, 20=IPF and 10 age and gender matched controls) were analyzed. Results were grouped in a non-bias hierarchal approach which clustered subjects with some potential overlap seen in IPF and controls as well as the observation of two groups of SSC. We observe remarkable differences between patients with different ILD sub-types based on un-biased statistical clustering (Figure). There are 4 sphingolipid clusters of interest. (1) In individuals with f-HP we observe a general loss of circulating sphingolipids specifically noted in the short chain isoforms 14-16. (2) In individuals with IPF we note an increase in long chain ceramides and sphingolipids, most notably in ceramide isoforms 22-26. We also observe 2 different clusters in SSc-ILD, (3) one group with distinct loss several sphingolipids, while the other cluster (4) is unique in a cluster of elevated sphingolipids. Conclusions: Though the complexity of sphingolipid metabolism in relationship to f-ILD is not fully understood, there appear to be distinct changes within their profile when compared to healthy controls and one another. This “signature” could be used to aid in more accurate diagnosis and management of individuals with f-ILD. Future research investigating if the changes in sphingolipid metabolism change following initiation of therapy, as well as leveraging the changes to further pursue disease mechanism and pathogenesis.

Background: 100% of cancer survivors who undergo thoracic radiation (TR) treatment are at risk of developing radiation-induced lung injury (RILI). Progressive RILI can lead to radiation-induced lung fibrosis (scar) a permanent diagnosis with no FDA-approved curative strategy. 90% of thoracic cancer survivors have CT-detectable RILI every year. Current techniques to limit RILI may make radiotherapy less effective at treating cancer. Our lab was the first to publish metabolomic findings from the exhaled breath condensate (EBC) of patients with diagnosed RILI. EBC is a natural matrix of the respiratory tract that represents the composition of the lung lining fluid. We reported that RILI is associated with global biosynthetic and bioenergetic metabolic defects including increased glycolysis and excess lactate production. Here, we hypothesized that targeting a key glycolytic enzyme, pyruvate kinase M2, will protect against radiation-induced myofibroblast differentiation. Radiation can transform lung fibroblasts into myofibroblasts. Myofibroblasts are the major scar-producing cells in fibrosis and thus the main effectors in the disease. Methods: In this study, primary human lung fibroblasts (pHLFs) were irradiated to induce myofibroblast differentiation and harvested after 5 days. We measured protein expression by western blot, and we performed targeted metabolomics analysis on cell lysates by liquid chromatography/tandem mass spectrometry. Glycolytic rate was measured by Seahorse assay. Results: Radiation markedly increases extracellular lactate (Fig1A). Radiation also induces alpha-smooth muscle actin, (α-SMA – myofibroblast marker), and other key proteins that make up scar (Fig1B). Radiation-induced myofibroblasts accelerate both basal and compensatory glycolysis shown (p = <0.0001) (Fig1C). Targeted metabolomics showed that major glycolytic intermediates accumulate significantly in radiation-induced myofibroblasts compared to non-irradiated controls namely fructose-1,6-bisphosphate (p = <0.0001), phosphoglycerate (p = <0.0001), and phosphoenolpyruvate (p = <0.0001) (Fig1D). Using both a genetic and pharmacologic inhibitor of pyruvate kinase M2, we blunted the final glycolysis reaction before irradiating the pHLFs. This inhibition significantly decreased radiation-induced myofibroblast differentiation (α-SMA, p = <0.0001) (Fig1E). Conclusions: We observed metabolic reprogramming associated with radiation-induced myofibroblast differentiation. Increased glycolysis and diversion to lactate were consistent with the metabolomic signature observed in patients who develop RILI and a mouse model of RILI (not shown here). Importantly, these results are comparable to metabolic reprogramming in cancer (Warburg). Targeting glycolysis disrupted a radiation-induced profibrotic phenotype.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive disease characterized by an excessive accumulation of extracellular matrix (ECM) that culminates in lung tissue scarring. Myofibroblasts are key players in ECM production, and fibroblasts are its precursors. Fibroblasts respond to mechanical stress such as stretch and stiffness through mechanosensors like PIEZO channels. Our lab has previously demonstrated an increase in PIEZO2 expression in lung tissue from patients with IPF. However, the specific downstream signaling pathways remain largely unexplored. This study aimed to elucidate the Piezo2 signaling pathways and evaluate the potential therapeutic impact in IPF. METHODS: Primary Human Lung Fibroblasts (HLFs) were cultured and treated with D-GsMTx4, a PIEZO2 inhibitor. After five days, proteins were extracted and analyzed using the Phospho Explorer Antibody Microarray (Full Moon Biosystems), following the manufacturer's protocol for sample preparation. Pathway enrichment analysis was conducted using String version 12.0. Heat maps were created with GraphPad Prism 10. RESULTS: Treatment of HLFs with D-GsMTx4 led to a notable decrease in phosphorylation levels across proteins within the mTOR and PI3K-Akt signaling pathways. Phospho-array analysis showed reductions in phosphorylation ranging from 30% to 62% across key targets, indicating effective pathway modulation by D-GsMTx4. Pathway enrichment analysis in STRING highlighted significant interactions among 45 critical proteins influenced by PIEZO2 inhibition, particularly within the mTOR signaling cascade. The analysis revealed modular clusters linked to fibrosis-related pathways, emphasizing mTOR as a central node in these regulatory networks. Furthermore, the clustering analysis underscored the specific impact of D-GsMTx4 on proteins known for their roles in cell growth, proliferation, and stress response within fibrotic processes. A comparative assessment of signal ratios for core pathway proteins (mTOR, 4E-BP1, AKT1, RAF1, IKKβ, eIF4E, and RPS6KB1) between treatment and control conditions underscored the reduction in phosphorylation status associated with PIEZO2 inhibition. CONCLUSIONS: These findings suggest that D-GsMTx4 selectively decreases activity along the mTOR axis and PI3/Akt signaling, thereby affecting downstream components that are crucial for cellular responses in fibrotic tissue remodeling. Piezo2 inhibition targeting these pathways could offer novel strategies to mitigate fibrosis in IPF, representing a promising direction for pulmonary fibrosis research. The findings are consistent with established processes in fibrosis and emphasize potential treatment avenues that require further experimental confirmation.

Rationale: Cigarette smoke (CS) is a potent stimulus that causes pulmonary inflammation, cell death and damage. CS is the primary cause of Chronic Obstructive Pulmonary Disease (COPD) in the United States which involves inflammation, emphysema and small airway remodeling. Specialized pro-resolving mediators (SPMs) are implicated in resolution biology where these endogenous lipids actively decrease inflammation and promote resolution. A newly discovered group of SPMs called the conjugates of tissue regeneration have the added novel effect of promoting tissue repair and regeneration. Here, we investigated whether Maresin Conjugate of Tissue Regeneration 3 (MCTR3) can protect against CS-induced epithelial damage and pro-inflammatory responses in primary human small airway epithelial cells (SAECs) by utilizing a highly translational model of differentiated SAECs at the air-liquid interface (ALI). Methods: SAECs were differentiated at the ALI for 4 weeks and exposed to CS for 30 mins/day for 4 days afterwards. CS was diluted with filtered air for an average exposure of 250-300 mg/m3 total particulate matter. 100 nM MCTR3 was added immediately after CS on days 1-4. On day 5, a FITC-dextran permeability assay was performed, media was collected for secreted IL-8 and MIF ELISAs, and the inserts were fixed for immunofluorescence staining of e-cadherin (adherens junction protein) and cleaved caspase 3 (apoptosis). 2-way ANOVA was used for statistics. Results: CS induced a 5-fold increase in IL-8 (p<0.001), which was reduced by 27% by MCTR3 treatment (p = 0.0196, Fig 1A). Similarly, CS induced a 4-fold increase in MIF (p = 0.0245), which was blunted back to baseline with MCTR3 (p = 0.0171, Fig 1B). CS caused a 3-fold increase (p = 0.0157) in the leak of FITC-dextran macromolecules across the epithelial barrier, and this was prevented by MCTR3 (p = 0.0163, Fig 1C). CS increased pro-apoptotic cleaved caspase 3 expression while decreasing adherens junction e-cadherin expression (Fig 1D, E). MCTR3 treatment prevented cleaved caspase 3 upregulation and rescued e-cadherin expression (Fig 1D, E). These results have been confirmed in 2 donors but representative data from one donor is shown. Conclusions: MCTR3 demonstrated its anti-inflammatory properties by decreasing CS-induced IL-8 and MIF cytokines which are both leukocyte chemoattractants. Notably, MCTR3 also prevented epithelial damage by decreasing apoptosis and maintaining SAEC barrier integrity after CS exposure. MCTR3 therefore has therapeutic potential for COPD since its properties may target uncontrolled inflammation along with damage to the epithelium (emphysema and small airway remodeling) experienced by patients.

Radiation-induced lung injury (RILI) is a consequence of therapeutic thoracic irradiation (TR) for many cancers, and there are no FDA-approved curative strategies. Studies report that 80% of patients who undergo TR will have CT-detectable interstitial lung abnormalities, and strategies to limit the risk of RILI may make radiotherapy less effective at treating cancer. Our lab and others have reported that lung tissue from patients with idiopathic pulmonary fibrosis (IPF) exhibits metabolic defects including increased glycolysis and lactate production. In this pilot study, we hypothesized that patients with radiation-induced lung damage will exhibit distinct changes in lung metabolism that may be associated with the incidence of fibrosis. Using liquid chromatogra-phy/tandem mass spectrometry to identify metabolic compounds, we analyzed exhaled breath condensate (EBC) in subjects with CT-confirmed lung lesions after TR for lung cancer, compared with healthy subjects, smokers, and cancer patients who had not yet received TR. The lung metabolomic profile of the irradiated group was significantly different from the three nonirradiated control groups, highlighted by increased levels of lactate. Pathway enrichment analysis revealed that EBC from the case patients exhibited concurrent alterations in lipid, amino acid, and carbohydrate energy metabolism associated with the energy-producing tricarboxylic acid (TCA) cycle. Radiation-induced glycolysis and diversion of lactate to the extracellular space suggests that pyru-vate, a precursor metabolite, converts to lactate rather than acetyl-CoA, which contributes to the TCA cycle. This TCA cycle defi-ciency may be compensated by these alternate energy sources to meet the metabolic demands of chronic wound repair. Using an "omics" approach to probe lung disease in a noninvasive manner could inform future mechanistic investigations and the de-velopment of novel therapeutic targets.NEW & NOTEWORTHY We report that exhaled breath condensate (EBC) identifies cellular metabolic dysregulation in patients with radiation-induced lung injury. In this pilot study, untargeted metabolomics revealed a striking metabolic signature in EBC from patients with radiation-induced lung fibrosis compared to patients with lung cancer, at-risk smokers, and healthy volunteers. Patients with radiation-induced fibrosis exhibit specific changes in tricarboxylic acid (TCA) cycle energy metabolism that may be required to support the increased energy demands of fibroproliferation.