Citations

Abstract Rationale The pathophysiology and clinical impact of liver injury in pulmonary hypertension (PH) is poorly understood. Liver injury in PH is often attributed to hepatic congestion, but some studies have failed to find strong associations with hemodynamics, suggesting alternative mechanisms. We sought to define the prevalence of liver injury and patterns of liver injury across the spectrum of PH and to determine whether patterns of liver injury were associated with clinical characteristics, hemodynamics and outcomes. Methods We performed an analysis of the PVDOMICS study, a multicenter, prospective, longitudinal cohort study that enrolled incident and prevalent patients with PH and disease comparators. The prevalence of liver injury, defined as aspartate or alanine aminotransferase (AST or ALT) or total bilirubin above the upper limit of normal, and different patterns of liver injury [hepatocellular (elevated AST or ALT with normal bilirubin), cholestatic (elevated bilirubin) and no liver injury] was defined among participants with known or suspected PH. Associations between patterns of liver injury with demographics, hemodynamics, right ventricular function, exercise capacity and quality of life (QoL) were assessed. We used Cox proportional hazards models adjusted for age and sex to assess the association of liver injury patterns with transplant-free survival. Results Liver injury was present in 27.8% (288/1036) of participants (19.0% cholestatic and 8.8% hepatocellular). Patients with liver injury were less likely to be female and have prevalent disease with differences in PH classification (Table 1). Patients with hepatocellular liver injury had a higher body mass index. A cholestatic liver injury pattern was most consistently associated with worse functional class, biventricular filling pressures, pulmonary vascular resistance, right ventricular function, natriuretic peptide elevation, exercise capacity, and REVEAL risk scores. Total bilirubin levels and right atrial pressure as a marker of systemic congestion were weakly correlated (r2=0.02, p < 0.0001). Compared to no liver injury, cholestatic liver injury was associated with an increased risk of death in age and sex adjusted models (HR 1.6, 95% CI 1.3-2.1, p < 0.001) but hepatocellular liver injury was not (p > 0.05). Conclusions Liver injury is common among patients with known or suspected PH. In contrast to a hepatocellular injury pattern, a cholestatic liver injury pattern was most consistently associated with worse hemodynamics, exercise capacity, QoL and survival. However, the weak relationship between right atrial pressure and bilirubin suggests that systemic congestion alone is unlikely to completely explain cholestatic liver injury. Further study is needed to uncouple the pathophysiological basis of cholestasis in PH. This abstract is funded by: NHLBI

Abstract Rationale Prior studies demonstrate pulmonary artery (PA) enlargement on computed tomography (CT) in pulmonary hypertension (PH). Some patients exhibit markedly enlarged or “aneurysmal” PA dilation, but its clinical relevance remains unclear. We aimed to characterize clinical characteristics, hemodynamics, and survival probability of patients with aneurysmal PA size. Methods Demographics, clinical characteristics, and CT imaging were obtained for individuals with group 1 or 2 PH and matched controls in the Pulmonary Vascular Disease Phenomics cohort. PA diameter was measured using Osirix software. PA diameter was categorized based on previously reported values as normal (<27 mm [females], <29 mm [males]), enlarged (27-39 mm [females], 29-39 mm [males]), or aneurysmal (≥40 mm). Outcomes included mean PA pressure (mPAP) on right heart catheterization (RHC) and time to death or heart/lung transplant. Associations with mortality and mPAP were evaluated using multivariable Cox and linear regression models, adjusting for age, sex, and BMI. Descriptive statistics are presented as median [IQR]. Results 690 subjects met the inclusion criteria for survival analysis (364 WSPH group 1 or matched comparator, 241 group 2 or comparator, 85 healthy control), with 594 undergoing RHC. PA diameter was normal in 163 participants, enlarged in 414, and aneurysmal in 113. Aneurysmal PA, compared with normal or enlarged PA, was associated with younger age (53 ± 14 vs 55 ± 16 and 59 ± 15 years, respectively; p < 0.001), non-White race (18.9% vs 8% and 17.2%; p = 0.034), pro-BNP (1950 ± 5708 vs 531 ±1651 and 110 ± 2258 pg/mL; p < 0.001), PH duration (6.78 ± 6.86 vs 2.24 ± 4.39 and 3.82 ± 5.47 years; p < 0.001), group 1 disease (77% vs 37% and 61%), and congenital heart disease (CHD)-PAH (10.6% vs 0.0% and 4.8%, p < 0.001). Aneurysmal PA subjects had higher mPAP (46 [36-57] vs 19 [16- 25] and 35 [28-47] mmHg, p < 0.001), pulmonary vascular resistance (PVR) (5.5 [3.6-9.4] vs 1.7 [1.0-2.3] and 4.1 [2.4-7.1] Woods units, p < 0.001), cardiac index (CI; 2.73 [2.34-3.29] vs 2.55 [2.13-2.98] and 2.52 [2.12-3.10] L/min/m2, p < 0.02) and likelihood of death or transplant (p < 0.001; Figure 1). Conclusion “Aneurysmal” PA was more common in younger, non-White subjects with longer WSPH group 1 disease duration, CHD, and higher pro-BNP levels. Aneurysmal PA was associated with elevated mPAP, PVR, and CI, and reduced likelihood of survival. These findings highlight the need to further elucidate the pathophysiology and prognostic implications of aneurysmal PA remodeling in PH. This abstract is funded by: NHLBI and the Pulmonary Hypertension Association

Abstract Background We recently reported a strong association between CT markers of pulmonary artery (PA) dilation and mean PA pressure (mPAP) in subjects with World Symposium on Pulmonary Hypertension (WSPH) group 1 and 2 pulmonary hypertension (PH). The relationship between PA size and mPAP in subjects with WSPH group 3 PH, in which parenchymal changes may influence PA dilation, remains uncertain. We sought to investigate this relationship in subjects with and without PH who have significant parenchymal pulmonary disease or hypoxia. Methods We measured PA diameter and PA:aorta (PA:A) ratio on CT for all subjects with WSPH group 3 PH, matched disease comparators, and healthy controls in the Pulmonary Vascular Disease Phenomics (PVDOMICS) cohort. PH and comparator subjects were categorized by group 3 etiology into obstructive (including chronic obstructive pulmonary disease [COPD] and cystic fibrosis), interstitial (including idiopathic pulmonary fibrosis, unspecified interstitial lung disease, combined pulmonary fibrosis and emphysema, and chronic hypersensitivity pneumonitis), and non-parenchymal (including obesity hypoventilation syndrome, obstructive sleep apnea, and extrinsic restrictive disease) subgroups. Primary outcome was mPAP on right heart catheterization (RHC). Association with mPAP was evaluated using multivariable linear regression adjusted for age, sex, and BMI. Results 339 subjects met inclusion criteria (101 WSPH group 3 PH, 153 disease comparators, 85 healthy controls). 83 subjects had obstructive disease, 125 had interstitial disease, and 46 had non-parenchymal disease. PA diameter and PA:A were associated with mPAP in all subjects and in each subgroup (p < 0.001; see figure 1). The association was notably stronger in the non-parenchymal group (ρ = 0.707 for diameter, 0.750 for PA:A) compared to obstructive (ρ = 0.566 for diameter, 0.529 for PA:A) and interstitial (ρ = 0.432 for diameter, 0.373 for PA:A) groups. Change in PA size between 25th and 75th percentile mPAP was lower in the interstitial group (3.88 mm, ratio 0.12) than in the obstructive (4.88 mm, ratio 0.17) and non-parenchymal (4.80 mm, ratio 0.19) groups (see figure 1). Conclusions PA size on CT is associated with mPAP in subjects with chronic lung disease with and without PH. This association is strongest in subjects without parenchymal lung disease but with features of hypercarbia or hypoxia alone. These data suggest that features of parenchymal lung disease in certain patients may limit expected PA dilation in response to elevated PA pressures. Further study is needed regarding the implications of these findings for early identification of PH in patients with chronic lung disease. This abstract is funded by: The NHLBI and Pulmonary Hypertension Association

Abstract Rationale Pulmonary hypertension (PH) is a heterogeneous condition classified into 5 WSPH groups but lacking detailed molecular distinction. The PVDOMICS cohort includes all PH groups alongside healthy and disease comparators, is deeply clinically phenotyped and has RNA sequencing data. Pseudogenes are conserved genome elements that due to sequence-defects cannot be translated into functional proteins. Some pseudogenes never-the-less produce RNA-transcripts which can regulate the expression of other, protein coding genes. Therefore, our study was focused on identifying differentially expressed pseudogene-transcripts within the dataset from the PVDOMICS study and gaining insights into their potential function. Methods Peripheral venous whole blood was collected from PVDOMICS participants [n = 525 PH patients; n = 312 controls without PH who were comparators (n = 256) or healthy (n = 56)]. RNA was isolated, RNA sequencing was performed. After quality control, differential transcript expression between PH and control groups was analyzed using the limma-voom pipeline, rSEA with adjustment for age, sex, race, smoking, WSPH group, latent factors and false discovery rate. We focused on a) transcripts expressed at increased levels (n = 218): the first 200 DEG (differentially-expressed-genes)-transcripts based on log2 difference plus increased DEG within the first 200 most significant p-values; and b) transcripts expressed at decreased levels (n = 292): last 200 DEG based on log2 difference plus decreased DEG within the first 200 most significant p-values. Results Six pseudogene transcripts were among the increased and 35 pseudogene transcripts were among the decreased DEG in the PH group compared to controls. Some of the differentially expressed pseudogenes (DEP) belonged to gene families, like the ribosomal protein family (7 DEP), mitochondria expressed transcripts (3 DEP), zinc finger proteins (2 DEP). Associations of pseudogenes with biological function or disease were extracted via GeneCards(R) and information from GWAS analysis. The DUSP8P5 pseudogene was among the DEP and is known for its association with left heart disease and hypertension; 2 additional DEP have known association with hypertension. Further known associations were with inhibitory or inflammatory immune processes (8 DEP), bone density (6 DEP), metabolism and diabetes (6 DEP), respiratory function or disease (4 DEP), erythrocyte biology (3 DEP), cancer (5 DEP), kidney disease (1 DEP), and demographic parameters like eye or hair color (2 DEP). Ten DEP had no known association with function or disease. Conclusions We identified pseudogene RNA-transcripts associated with PH across WSPH groups. The local vs. systemic effects of these pseudogene transcripts on PH remain to be determined and regulators of pseudogene-transcript expression (e.g. environmental exposures) identified. This abstract is funded by: 1 X01 HL148611 (TopMed), PVDOMICS NHLBI, PVDOMICS Pulmonary Hypertension Association, HT9425-25-1-0533 (GG), 1R21OH012955-01 (GG)

Abstract Background Single breath diffusing capacity of the lung for carbon monoxide (DLCO) remains the most widely used test to assess pulmonary gas exchange. GLI DLCO reference equations are based on a larger, more diverse dataset and offer age-specific and internally consistent reference values. However, their impact on interpretation in a real-world population remains uncertain. Research Question Describe the direction and magnitude of DLCO interpretation changes resulting from our multicenter PFT laboratory transition from Miller to GLI reference equations. Study Design and Methods In this multicenter cross-sectional study, we reanalyzed 88,060 DLCO measurements in a large pulmonary function laboratory database performed between 2016 and 2022 using GLI and Miller reference equations, stratified by self-reported race and ethnicity. Baseline patient characteristics and lung function measurements were summarized with continuous characteristics as median (IQR) and categorical variables as n (%). Kruskal-Wallis test was used to evaluate the difference between GLI and Miller DLCO % predicted difference between races, and two-by-two and four-by-four tables were generated to summarize the proportion of concordance and discordance of GLI to Miller severity levels. A repeated measures multivariable logistic regression was used to evaluate discordance between GLI and Miller severity and patient characteristics (age, sex, race, and BMI). Results We included 63,357 individuals with 88,060 PFT visits; 91.6% (58,076) identified as Caucasian, 5.2% (3,262) as Black, 1.4 % (895) as Asian, and 1.8% (1,113) as mixed/other race. Fifty-one percent were female with an median age of 64 years (range: 5- 85). 7.4% (6509) of DLCO results were discordant, with 0.5% (n = 439) abnormal by GLI only, and 6.9% (n = 6070) abnormal by Miller only. Compared to the Miller, GLI interpretations were associated with a greater shift toward normal interpretations. Younger age, lower BMI, female sex, and being of NE Asian, SE Asian, or Other/mixed race compared to Caucasian were associated with higher odds of discordance in DLCO severity/normal classification. Interpretation The use of GLI reference equations substantially alters the interpretation of DLCO results, shifting a meaningful subset of patients from abnormal to normal classification. While GLI reference equations offer more comprehensive, population-based approach, it is crucial to place these interpretations within the context of the patient presentation until further investigations more clearly define the clinical implications of these transitions. This abstract is funded by: none

Abstract Introduction The inhalation of aerosolized therapeutics is a promising strategy for the targeted delivery of drugs and biologics to the lungs. We have previously demonstrated the efficacy and feasibility of nebulized exosomes using a platelet-derived exosome product (PEP: Rion, Inc.), which significantly attenuated emphysema in a murine model (PMID: 38325750). However, the PEP aerosol’s physical properties—such as size, tissue deposition, anatomic location, and stability remain incompletely characterized. To address this, we utilized a cascade impactor to define aerosol particle size and deposition patterns and employed immunofluorescence staining to assess the regional and anatomic localization of nebulized PEP within the murine lung. Methods PEP particle size and tissue deposition were determined by passing the aerosol through a Marple cascade impactor (CI, DSI), which was connected to an inhalation tower (DSI). PEP aerosols were generated by a vibrating mesh nebulizer (Aerogen, Cat No AG-AL1100) at a controlled velocity and humidity and delivered to the CI at a flow rate of 2L/min. Aerosols were separated by size and allowed to deposit at the corresponding stages as they passed through the pinholes of the stage, by design. The stages were weighed after nebulization, and the percentage of aerosol deposition was calculated to simulate the human respiratory tree, according to the manufacturer’s specifications. In parallel, fluorescence DiR-labeled PEP was nebulized to spontaneously breathing mice, and confocal microscopy was used to determine intrapulmonary distribution. Results A total of 2.25 mL of PEP reconstituted in 0.9% normal saline (11.4 mg/mL final concentration) was nebulized for 35 minutes at a flow rate of 2 L/min, yielding 3.5 mg of total aerosol deposition, which is 13.6% of the total PEP mass (25.65mg) being nebulized, across the CI stages. Of this, 74% of the aerosolized particles with a particle size ≤ 1µm and deposited in Stages 7, 8 & F, corresponding to the deep regions of the lungs, specifically regions beyond the alveolar ducts and within the alveoli. In vivo 3D confocal imaging corroborated these findings, revealing abundant DiR-labeled PEP deep into the alveolar space within the peripheral regions of all lung lobes. Conclusion The study result demonstrates the feasibility and capability of delivering aerosolized PEP deeply into the alveolar regions of the lung at the given pressure, humidity, flow rate and nebulizer settings. This study establishes a methodological framework for characterizing and optimizing aerosolized exosome therapeutics for precise, region-specific delivery within the respiratory tract. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Abstract Rationale Alpha1-antitrypsin (AAT; gene name SERPINA1) is a critical antiprotease that protects the lung from neutrophil elastase-mediated injury. AAT deficiency predisposes to emphysema, which is further exacerbated by cigarette smoke (CS) exposure. We previously reported (PMID: 38325750) that a nebulized human platelet-derived exosome product (PEP) attenuated CS-induced emphysema in C57BL/6 mice. Given the presence of AAT protein in these platelet-derived exosomes (identified by proteomics), we hypothesized that PEP nebulization would significantly attenuate both spontaneous and CS-induced emphysema in SERPINA1 knockout (KO) mice. Methods Transgenic SERPINA1 KO C57BL/6 mice (both sexes) exposed to CS (10 cigarettes/day and 5 days/week) for 2 months were then randomized to receive nebulized PEP or 0.9% saline (placebo) twice weekly for 4 additional weeks, with continued CS exposure. PEP was reconstituted in 0.9% saline (22.7mg/mL; ∼3 × 10¹0 vesicles/mL) and administered via pressure- and humidity-controlled nose-only inhalation tower (2.25 mL of PEP or saline over 35 minutes). Lung static compliance (Cst), cutaneous O2 saturation, mean linear intercepts (Lm), and plethysmographic parameters were measured during and after the study. Results Histological analysis of the lung tissue sections revealed that PEP significantly attenuated the CS-induced increase in alveolar size, as determined by measurement of the mean linear intercepts (Lm, p < 0.001). Lung static compliance (Cst), which was increased in CS-exposed mice, was also significantly decreased in the PEP-treated group (p < 0.001) compared to placebo in the male and female combined group. Functional respiratory parameters further supported the protective effect of PEP in this AAT-deficient model. In female mice, PEP nebulization significantly improved several CS-impaired pulmonary function parameters, including minute volume (MV, p = 0.024), the flow at the point 50% of tidal volume is expired (EF50, p = 0.021), Peak Inspiratory Flow (PIF, p = 0.009), Peak Expiratory Flow (PEF, p = 0.018), and O2 saturation (p = 0.035). However, these improvements were not observed in male mice. Conclusions These findings extend prior observations and underscore the therapeutic potential of nebulized AAT-enriched PEP directly to the alveolar region, effectively attenuating injury and improving pulmonary function in AAT deficiency-related emphysema. This abstract is funded by: Department of Defense, Mayo Clinic Funding, The Young and Middle-aged Medical High-end Talents in Chongqing Program

Abstract Introduction We previously demonstrated that nebulized platelet-derived exosome product (PEP) alleviates cigarette smoke (CS)-induced murine emphysema. To elucidate the underlying molecular mechanisms, we conducted single-nucleus RNA sequencing of lung tissue, which initially revealed a shift in the cell source for TNF signaling (a critical regulator of inflammation in COPD lungs) from innate lymphoid cells (ILC) to alveolar macrophages (aMac) after chronic CS exposure. In this study, we conducted a more in-depth analysis of cellular communication to determine the mechanism by which PEP modulates CS-induced TNF signaling in the lung microenvironment. Methods Single-cell transcriptomics and cell-cell communication analyses were performed between CS-exposed placebo control lungs vs. sham control lungs and CS-exposed PEP-treated vs. placebo control lungs using R packages, including Seurat, CellChat, Patchwork, Future, and NMF. A comparative analysis of ligand-receptor interactions across these datasets was conducted using CellChat to infer intercellular signaling networks. Results Single-nuclear RNA-seq analysis revealed that the Thy1-ITGAX_ITGB2 signaling pathway was significantly upregulated by CS, acting as the adhesive and activating bridge that facilitates the switch in TNF production from ILC to aMac. Thy1 is a GPI-anchored ligand expressed on ILCs, and ITGAX_ITGB2 is its cognate receptor on aMacs. Concurrently, Ptprc-Mrc1 signaling, an inhibitory pathway in ILC-aMac crosstalk, was downregulated, contributing to sustained aMac activation by CS. Furthermore, analysis of aMac-aMac interactions revealed upregulated GRN/Sort1 signaling, which promotes the internalization of progranulin, an anti-inflammatory glycoprotein that competes for TNF/TNFR signaling. Ptprc-Mrc1 signaling was also downregulated within the aMac autocrine/paracrine network, further perpetuating a pro-inflammatory state. In contrast, treatment with PEP downregulated Thy1-ITGAX_ITGB2 signaling and upregulated Lamc1-Cd44 signaling, a hallmark of aMac deactivation, thereby inducing a phenotypic switch in inflammatory macrophages toward a reparative M2 state, promoting the resolution of CS-induced chronic inflammation. Conclusions These data suggest that the transition of TNF production from ILCs to aMacs under chronic CS stress is driven by increased Thy1-ITGAX_ITGB2 interactions and decreased Ptprc-Mrc1 signaling between ILCs and aMacs crosstalk within the local tissue. Enhancing the crosstalk between Lamc1-Cd44 and Ptprc-Mrc1 may suppress TNF production and attenuate CS induced aMac activation. Targeting these pathways, as demonstrated by nebulized PEP treatment, holds therapeutic potential to deactivate inflamed aMacs and promote homeostasis in the inflamed lung. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Abstract Rationale Type-1 inflammation in asthma leads to significant airway level changes independent of Type-2 inflammation and may underlie the severity seen in combined Type1/2 high disease. As IFNs can cause airway epithelial injury and impaired repair, we applied a novel interpretable machine learning algorithm, SLIDE, to analyze the transcriptome of airway epithelial brushings of mild-moderate (MMA) and severe asthma (SA) participants in the Immune Mechanisms in Severe Asthma (IMSA) cohort to identify latent/hidden factors of disease that associate with disease severity. Methods The expression of genes identified by SLIDE as part of a co-expression program was assessed in the full IMSA cohort by severity and in an independent cohort of the NHLBI Severe Asthma Research Program (SARP). We also examined associations of these genes with clinical asthma outcomes in both cohorts. Results SLIDE identified aberrant increased expression of membrane-tethered mucin genes, MUC1 and MUC4. MUC1 appeared in each of 5 significant latent factors associated with greater severity and MUC4 in 2/5. Standard DGE analysis also identified the gene CEACAM5 as a predictor of severity. Pathway analysis of latent factor genes revealed airway keratinization which may contribute to airway remodeling and worsening obstruction over time. Expression of MUC1, MUC4, and CEACAM5 were increased in SA compared to HC in both the full IMSA and separate SARP cohorts (p < 0.001 for all genes in both cohorts). Expression in MMA was consistently lower than in SA in both cohorts, reaching significance in IMSA (p < 0.01) but not consistently in SARP (MUC1 p = 0.04, MUC4 p = 0.27, CEACAM5 p = 0.27). Protein analysis of endobronchial biopsies by immunofluorescence staining showed a trend towards increased MUC1 protein content with evidence of secretion. MUC1, MUC4 and CEACAM5 were negatively correlated with FEV1 % predicted in both cohorts (IMSA: MUC1 r=-0.47, p < 0.01, MUC4 r=-0.47, p < 0.01, CEACAM5 r=-0.62, p < 0.01; SARP: MUC1 r=-0.19, p = 0.05, MUC4 r=-0.29, p < 0.01, CEACAM5 r=-0.51, p < 0.01). All three genes positively correlated with prospective exacerbations in both cohorts (IMSA: MUC1 r = 0.345, p = 0.01, MUC4 r = 0.216, p = 0.08, CEACAM5 r = 0.368, p < 0.01; SARP: MUC1 r = 0.22, p = 0.02, MUC4 r = 0.32, p < 0.01, CEACAM5 r = 0.38, p < 0.01). Conclusions The expression of MUC1, MUC4 and CEACAM5 increased with asthma severity in the airway epithelium and correlated with both prospective exacerbations and obstruction. As pathway analysis of genes in the significant latent factors were associated with airway keratinization aberrant expression of these mucins (with known signaling functions) may contribute to maladaptive repair and airway remodeling that drives obstruction over time, contributing to worse asthma outcomes. This abstract is funded by: NIH

Importance Rectovaginal fistulas (RVFs) are challenging to treat, often requiring multiple surgical interventions. Research into novel therapies is limited by the lack of a sizeable and representative animal model. Given the similarities in porcine and human vaginal and rectal tissue, pigs are an ideal species for translation to human patients. Objective The objective of this study was to develop a feasible and reproducible RVF model in pigs. Study Design Six female pigs underwent RVF creation and were divided into 3 groups according to the following methods: (1) placement of 1-inch disc magnets across the rectovaginal septum, (2) a 1-inch round defect created using electrocautery and placement of a 1-inch stainless-steel gauge stent, (3) a 1-inch round defect using electrocautery without subsequent stenting. Fistulas were evaluated at 6 weeks with measurements and photographic documentation. The animals were euthanized between 12 and 14 weeks. The primary outcome of this study was fistula patency. Secondary outcomes were fistula dimension and histologic appearance of the fistula tract. Results All pigs did well with no infection or complications related to fistula creation. Both surgical methods reliably resulted in a patent fistula at 14 weeks: cautery alone and cautery with gauge stenting. Only 1 patent fistula in the magnet group was noted at the time of euthanasia. All patent fistulas showed histologic evidence of complete epithelialization of the fistula tract. Conclusions This study demonstrates 3 safe and feasible methods for RVF creation in pigs, with 2 methods showing increased reliability.