Citations

Abstract Rationale Pulmonary hypertension (PH) is classified into five groups. Diagnosis requires multiple tests delaying treatment. This study compared patient and diagnostic characteristics for pulmonary arterial hypertension (PAH; Group 1 PH), Group 2-5 PH, and non-PH using machine-learning in an Electronic Health Record (EHR) dataset. This is novel as prior studies use claims data which may lack hemodynamic parameters. Methods This study analyzed adult patient records from a HIPPA-complaint EHR dataset from Mayo Clinic (2015-2024). Cohort definitions: PAH: right heart catheterization (RHC) data [mPAP>20 mmHg, PVR>2 WU, PCWP≤15 mmHg]; ≥2 PAH ICD diagnosis codes or positive natural language processing (NLP) sentiments separated by ≥ 30 days; and use of at least one PAH medication within six months after index date. Group 2-5 PH: RHC (mPAP>20mmHg) and ICD codes or positive NLP sentiment separated by ≥ 30 days for corresponding PH subtype. Non-PH: mPAP ≤ 20 mmHg and/or TRV ≤ 2.8 m/s. Index was the earliest NLP or ICD for PAH, earliest RHC for group 2-5 PH and most recent TRV/ mPAP for the non-PH cohort. Clinical variables were analyzed one-year before study index acknowledging data missingness as a limitation of retrospective dataset studies. Results 593 PAH, 817 Group 2-5 PH (67% with group 2 PH), and 11,600 non-PH patients (1:10 propensity-matched) were identified. Age, race, and symptoms were similar across cohorts. PAH had the highest proportion of females (60%) [Group 2-5 PH (50%) and non-PH cohort (53%)]. Clinical characteristic comparison is presented in Table 1. Comorbidities differed across cohorts. RHC: As expected, compared to Group 2-5, the PAH cohort had higher mPAP and PVR and lower PCWP. confirming a distinctly pre-capillary profile. ECHO: Compared to Group 2-5 PH, the PAH cohort had more pronounced right ventricular (RV) pressure overload and dysfunction and more impaired RV longitudinal strain, while TAPSE was similar. At the same time, left heart structure was less affected in PAH compared to Group 2-5. Among laboratory tests, serum creatinine was higher in Group 2-5 and ANA was higher in PAH cohort. Compared to the no-PH cohort, PAH and Group 2-5 cohorts exhibited clinically non-normal RHC and ECHO findings. Conclusion Although symptoms and demographic characteristics were similar among patients with and without PAH, unique RHC, ECHO, laboratory profiles and comorbidities differentiated PAH patients. Screening techniques using these distinct profiles could be considered to facilitate earlier diagnosis and treatment of PAH in clinical settings. This abstract is funded by: Merck & Co., Inc., Rahway, NJ, USA

Abstract Rationale Pulmonary arterial hypertension (PAH) is a rare and progressive disease, where the guideline-recommended diagnostic confirmation is via right heart catheterization (RHC). However, due to non-specific symptomology, patients often experience delays in diagnosis that potentially impact disease outcomes. We sought to develop a machine learning (ML) based approach for early and appropriate detection of PAH. Methods Three cohorts were derived from Mayo Clinic electronic health record (EHR): confirmed PAH (N = 1,169), No PH (N = 11,600) and Other PH (Group 2-5 PH, N = 817), based on 2022 ESC/ERS guidelines. The study index was defined as the earliest diagnosis dates for PH cohorts (PAH & Other PH) or the latest PH negative echocardiogram (ECHO) or RHC for the No-PH cohort. Patients included in the study had at least one symptom (dyspnea, fatigue, syncope, chest pain, or lower extremity swelling) within one year prior to the study index. A two-step sequential ML modelling strategy was applied: Step 1 differentiated PH (PAH + Other PH) from No PH and step 2 differentiated PAH from Other PH. Models were trained using a combination of structured data, unstructured clinical notes to detect symptoms using natural language processing and ECG waveforms captured one year prior to study index. Models were tested both with and without ECHO parameters. SHAP (SHapley Additive explanations) values were used to determine important features contributing to models (without ECG waveforms). Results Models including ECHO parameters performed better compared to those without (Figure 1). The top feature impacting model without ECHO parameters was dyspnea in the preceding three months followed by heart failure and systolic blood pressure for the PH vs no PH; and T wave peak in V1 and ANA level in PAH vs other PH cohort. The top feature impacting model with ECHO was tricuspid regurgitation velocity followed by heart failure and right atrial pressure for the PH vs no PH; and mean blood pressure and left ventricular diameter in the PAH vs other PH cohort. Figure 1: Model performance in the two-step sequential approach PR-AUC=Precision Recall-Area Under Curve Conclusion This study demonstrates that ML models can aid in the identification of PAH in symptomatic patient populations. While ECG can identify patients for further testing, ECHO can further increase the accuracy and the need to refer patients to RHC for confirming diagnosis of PAH. This abstract is funded by: Merck & Co., Inc., Rahway, NJ, USA

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 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 Oscillometry measures the mechanical properties of the respiratory system using the response to pressure signals superimposed on tidal breathing. The JaegerTM IOS device generates “pulse packets” of harmonic frequencies between 5 and 35 Hz in 5 Hz intervals, while the Resmon ProTM V3 FOT and Thorasys TremofloTM C-100 employ prime pseudorandom sine waveforms between 5-19 Hz and 5-37 Hz, respectively. Prior studies have raised concerns about harmonic distortion with the IOS technique, but a direct comparison of reproducibility using current devices has not been performed. Methods Healthy volunteers with normal spirometry performed weekly oscillometry following ERS technical standards using JaegerTM and ResmonTM or ThorasysTM oscillometers in random order for 10 consecutive weeks. Z scores were calculated using Berger and Oostveen normative equations. Means, pooled within-subject distributions and interquartile range, standard error (SEM), coefficient of variation (CVw%), absolute and relative minimal detectable change (MDC, MDC%), and between device and subject intraclass correlation coefficients (ICC) were calculated for R5, R10/R11, R20/R19, R5-R20/R5-R19, X5, AX, and Fres and compared. Results Fifteen participants completed 10 weekly measurement sessions with JaegerTM and ResmonTM and twelve with JaegerTM and ThorasysTM. One subject did not complete the ResmonTM analysis due to intolerance; another subject only completed three weeks of ThorasysTM prior to work assignment change. The majority (65%) were female with mean (SD) age of 37.7 (10.2) years and BMI 27 (5) kg/m2. Rrs reproducibility was excellent across devices (ICC > 0.89), with more variability for X5, AX, and Fres (ICC 0.66 - 0.93) and little difference between devices (Figure 1). Small, systematic increases in mean Rrs were seen with JaegerTM compared to ResmonTM (5-10%, p < 0.008) and TremofloTM (5-12%, p < 0.02), with higher mean AX vs ResmonTM (44%, p = 0.002) and lower Fres vs TremofloTM (19%, p = 0.02). There was high ICC (0.97-0.99) across ResmonTM and TremofloTM Rrs comparisons with minimal bias. Intra-subject reproducibility was similar between devices. MDC% was 22.7-39.4% for Rrs values, 52.7-70.8% for X5, and 131-142% for AX. Conclusions JaegerTM, ResmonTM, and ThorasysTM oscillometers performed similarly in healthy adults over 10 weeks, with small but systematic measurement differences seen with the JaegerTM device. Rrs and Fres values demonstrated the greatest reproducibility both within and between subjects. Reactance measurements had less between-device agreement and higher MDC. Rrs, X5, and AX changes of up to 40%, 70%, and 140% over time may be due to normal subject and measurement variation. This abstract is funded by: 2024 Pulmonary, Critical Care, and Sleep Medicine Schuen Family Innovation Award

Abstract Rationale The governing mechanisms of lung regeneration are still poorly understood. Studies have observed the lung’s regenerative capacity using the pneumonectomy (PNX) model, where removal of the left lung stimulates compensatory growth and neoalveolarization. However, this process is inhibited if a physical object is used to replace the extracted tissue, demonstrating that the lung can sense the available space and regenerate in response. The mechanism of this mechanosensing is unknown. As outermost tissue layer of the lung, the pulmonary mesothelium is well situated to sense the surrounding environment. We and others have hypothesized that the mesothelium can become activated in cases of injury or regeneration, but this has not been fully explored. Understanding the mechanisms controlling neoalveolarizaiton may be key to unlocking the lung’s natural regenerative capacity in humans. Methods & Results In this study, we examined the location and fate of the mesothelium during regeneration using fluorescent lineage tracing and the PNX model. Specifically, we used the mesothelial-specific Wt1CreERT2 crossed with a lox-stop-lox-RFP reporter (Wt1(CreERT2/+);LSL-RFP(+/-)). Following PNX, we collected the lineage-labeled cells along with the CD326+ and CD326-/CD45-/CD31- populations and performed scRNA-sequencing. As quantified by flow-cytometry, the number of RFP+ cells increased following PNX, indicating proliferation of the mesothelium. Clustering analysis elucidated the emergence of 3 activated clusters of mesothelium following PNX, representing 12.9% of the RFP+ population in Sham and 66% in PNX. These activated cells exhibited decreased expression of mesothelial hallmarks and increased expression of extracellular matrix and epithelial-to-mesenchymal transition genes. Using immunofluorescence and confocal microscopy, we observed lineage-labeled mesothelium deep within the parenchyma after PNX. We observed none or singular invaded mesothelium in no-procedure control animals and only minimal occurrence in animals exposed to Sham surgery. Compared to Sham controls, PNX post-surgical (D)ay 3, D7, and D14 had 8.5, 32.7, and 9.2-fold more cells, respectively, in the parenchyma. Moreover, in aged (18 month) mice, where regeneration is known to be significantly reduced, we likewise observed minimal mesothelial invasion following PNX. These cells were often found near or directly wrapping around progenitors of the alveolar epithelium, AT2 cells, whose proliferation is essential to neoalveolarization. Using time-lapse microscopy of precision cut lung slices, we observed the invading mesothelial cells to be highly dynamic, sending out multiple transient protrusions and interacting with neighboring cells. Conclusion Together these data support strong and potentially functional interactions between activated and invading mesothelial cells and lung regeneration following PNX. This abstract is funded by: NIH

Abstract Rationale Normal lung aging is characterized by progressive structural and functional decline that reduces respiratory efficiency. Aging is associated with excessive reactive oxygen species production and impaired homeostatic pathways that maintain mitochondrial quality control, involving mitophagy (removal of damaged mitochondria) and mitochondrial biogenesis (generation of new mitochondria). In human airway smooth muscle (hASM), reduced mitochondrial quality control can lead to the accumulation of damaged mitochondria, compromising ATP production and decreasing cellular viability. Maintaining healthy mitochondria involves a balance between mitophagy and mitochondrial biogenesis. A key regulator of this balance is PARIS, normally a target of the PINK1/pParkinS65 mitophagy pathway. PARIS transcriptionally inhibits PGC1α and NRF2 expression, key regulators of mitochondrial biogenesis and redox homeostasis. Furthermore, NRF2 can upregulate PINK1 expression, enhancing mitophagy. Therefore, we hypothesize that aging involves unmitigated mitochondrial dysfunction due to accumulation of PARIS and an imbalance between the removal of damaged mitochondria (mitophagy) and mitochondrial biogenesis. Methods hASM cells were dissociated from bronchiolar tissue samples from patients with no history of smoking or respiratory diseases and cultured (≤3 passages). Prior to experimentation, hASM cells from young (≤40 yrs) and old (≥70 yrs) patients (female and male) were serum deprived and phenotyped. In both age groups, targeted siRNA-mediated knockdown of PARIS was also performed. The extent of mitochondrial damage in hASM cells was determined by changes in mitochondrial membrane potential (Ψm) using TMRM (250 nM), and the extent of co-labeling of MitoTracker Red (200 nM) and CellLight GFP. Mitochondrial turnover was quantified by confocal imaging of hASM cells transfected with pMitoTimer. Mitophagy was determined by PINK1 recruitment to mitochondria, pParkinS65 and ubiquitin (pUbS65) phosphorylation. Total PARIS and pParkinS65-ubiquitinated PARIS (by immunoprecipitation) levels were determined. Mitochondrial biogenesis was determined by PGC1α and NRF2 mRNA and protein expression and mitochondrial DNA (mtDNA) copy number. Results In hASM cells from older patients, there was extensive mitochondrial damage, decreased PINK1 recruitment to mitochondria and decreased pParkinS65 and pUbS65 phosphorylation. Total PARIS accumulation increased due to decreased PARIS ubiquitination. Also, PGC1α and NRF2 expression, and mtDNA copy number decreased, likely due to PARIS-mediated inhibition. Knockdown of PARIS increased PGC1α and NRF2 expression, thereby promoting mitochondrial biogenesis and NRF2-mediated antioxidant responses and increasing PINK1 expression, thereby activating mitophagy. Conclusion Aging results in impaired mitochondrial quality control in hASM cells. The PARIS-NRF2 axis presents a promising target for therapeutic intervention to mitigate oxidative damage, maintain mitochondrial homeostasis and improve lung function with aging. This abstract is funded by: HL157984 (GCS) and AG44615 (GCS)

Abstract Rationale Pulmonary Fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), primarily collagen I, leading to progressive lung scarring and loss of function. Many anti-fibrotic compounds have been found to attenuate collagen deposition. However, identifying compounds that stimulate collagen I degradation, a necessary step in fibrosis clearance, is less common. To identify such compounds, we developed in vitro and ex vivo assays to quantify extracellular collagen I and collagen I degradation products. Methods Primary lung fibroblasts (passage 1-3, N = 5) from patients with idiopathic pulmonary fibrosis (IPF) were cultured in DMEM/F12 with 2% FBS, 2 ng/mL TGF-β, and 50 µg/mL ascorbic acid for 72 hours to generate collagen-rich cell-derived ECM. Cells were then treated with test compounds for 24 hours in the same media. Nuclear labeling with Hoechst 33342 was used to quantify cell numbers by automated microscopy. Decellularization was then performed with a solution containing 20 mM ammonium hydroxide and 0.5% Triton X-100. Cell-derived ECM was then fixed, stained with anti-Collagen I antibody, and quantified by automated microscopy. Collagen I degradation was further assessed by measuring type I collagen C-telopeptide (ICTP) via ELISA in cell culture media. To investigate the role of matrix metalloproteinase-13 (MMP-13) in collagen degradation, we used T26-C, a selective MMP-13 inhibitor, or transfected cells with MMP-13 siRNA. Ex vivo validation of collagen-degrading activity was conducted by measuring type I collagen N-terminal telopeptide (NTX-I) in the culture media of human precision-cut lung slices (PCLS, N = 3) generated from upper and lower lobes of IPF tissue and stimulated with select compounds. Results Thirty-four candidate compounds (metabolic, epigenetic, and cAMP modulators) were tested for their ability to reduce collagen I levels in cell-derived ECM. Six hits and two positive controls were then validated using ICTP ELISAs. In total five compounds were found to stimulate collagen degradation: EGCG (1 μM), Nintedanib (0.25 μM), SB431542 (1 μM), and two dopamine receptor agonists, A68930 (10 μM) and Dihydrexidine (10 µM). MMP-13 inhibition or knockdown prevented collagen degradation by all five compounds. In human IPF PCLS, collagen degradation was confirmed in response to EGCG, Nintedanib and SB431542 but not Dihydrexidine, with much greater responses seen in PCLS from upper lobes than lower lobes. Conclusion Collagen degradation can be stimulated in cultured fibroblasts and IPF PCLS. These systems will assist in uncovering mechanisms and regulators of collagen degradation and may pave the way to better fibrosis-resolving therapies. This abstract is funded by: NIH U01 HL152967

Abstract Rationale Respiratory disease is a major cause of mortality in Down syndrome (DS; trisomy 21, T21). DS is considered a progeroid syndrome, with elevated cellular senescence (SEN) and oxidative stress. Because SEN shapes organogenesis, its dysregulation may impair lung morphogenesis. We previously found SEN/SASP (senescence-associated secretory phenotype) abnormalities in prenatal human T21 lungs. Here, we test whether these alterations persist and change after birth. Methods We analyzed human lung single-nuclear RNA (snRNA-seq) datasets (prenatal and postnatal) to quantify epithelial and mesenchymal SEN/SASP signatures. Sex- and age-matched T21 and non-T21 prenatal and postnatal lungs were studied; fibroblasts were isolated as prenatal human lung fibroblasts (HFLF) and postnatal human lung fibroblasts (HLF). SEN/SASP were measured by RT-qPCR and immunofluorescence (IF) in tissue and HFLF/HLF. HFLF/HLF were assayed for SA-β-gal, oxidative stress (CellROX/MitoSOX), and Ca²+ dynamics (Fura-2 AM). 48h treatments with tunicamycin (1µM, SEN inducer), 4-phenylbutyrate (4-PBA, 2 mM, ER Stress inhibitor), or pyrroloquinoline quinone (PQQ, 20 μM, antioxidant) were performed on T21 and non-T21 HFLF/HLF. Conditioned media (CM) were collected after 48h from untreated cultures and then applied to cells of the opposite genotype for 24, 48, and 72h. Results Overall SEN profile was similar in epithelium and mesenchyme across ages, but pathway signatures differed. Epithelium shows an inflammatory SASP, while mesenchyme shows a less-inflammatory, remodeling/growth-factor SASP. These lineage patterns were more evident postnatally. Whole-tissue IF showed increased γ-H2AX and p21 in T21 vs non-T21 both prenatally and postnatally (p < 0.05). At baseline, T21 HFLF/HLF had higher CDKN1A, CDKN2A, TP53, CDKN2B, and SERPINE1 (p < 0.05; n = 5-6), with increased γ-H2AX/p21 IF (p < 0.01; n = 6), SA-β-gal (p < 0.05; n = 5-6), and ER/mitochondrial stress (p < 0.05; n = 5-6). Fura-2 AM showed greater Ca²+ release in T21 HFLF/HLF at baseline and after acetylcholine (10 μM, p < 0.01; n = 5). Mitochondrial-morphology genes (FIS1, DNM1L, MFN1) were elevated in T21 HFLF/HLF (p < 0.05; n = 6). In T21 HFLF, 4-PBA and PQQ each reduced SA-β-gal (p < 0.01; n = 6). In postnatal HLF, PQQ had the stronger effect (p < 0.05; n = 5). T21 CM induced progressively greater SEN/SASP over 24/48/72h in non-T21 cells (p < 0.05; n = 5-6), while non-T21 CM partially rescued T21-HLF phenotypes at 24h only (p < 0.05; n = 5-6). Conclusion These data indicate that lung senescence in T21 initiates in utero and intensifies after birth and reflects lineage-specific pathways. ER-stress-related mechanisms are more prominent prenatally, while mitochondrial/redox mechanisms are predominant postnatally. This evidence suggests age and lineage-dependent drivers of senescence that may help explain DS lung disease and highlight potential new mechanistic targets. This abstract is funded by: NIH/NHLBI

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