Tissue Damping Research Articles

Adam19 Deficiency Impacts Pulmonary Function: Human GWAS Follow-up in a Mouse Knockout Model

PurposeOver 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function.MethodsWe created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent.ResultsContrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways.ConclusionOur murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.

Aerobic physical training reduces severe asthma phenotype involving kinins pathway.

Aerobic physical training (APT) reduces eosinophilic airway inflammation, but its effects and mechanisms in severe asthma remain unknown. An in vitro study employing key cells involved in the pathogenesis of severe asthma, such as freshly isolated human eosinophils, neutrophils, and bronchial epithelial cell lineage (BEAS-2B) and lung fibroblasts (MRC-5 cells), was conducted. Additionally, an in vivo study using male C57Bl/6 mice, including Control (Co; n = 10), Trained (Exe; n = 10), house dust mite (HDM; n = 10), and HDM + Trained (HDM + Exe; n = 10) groups, was carried out, with APT performed at moderate intensity, 5x/week, for 4 weeks. HDM and bradykinin, either alone or in combination, induced hyperactivation in human neutrophils, eosinophils, BEAS-2B, and MRC-5 cells. In contrast, IL-10, the primary anti-inflammatory molecule released during APT, inhibited these inflammatory effects, as evidenced by the suppression of numerous cytokines and reduced mRNA expression of the B1 receptor and ACE-2. The in vivo study demonstrated that APT decreased bronchoalveolar lavage levels of bradykinin, IL-1β, IL-4, IL-5, IL-17, IL-33, TNF-α, and IL-13, while increasing levels of IL-10, klotho, and IL-1RA. APT reduced the accumulation of polymorphonuclear cells, lymphocytes, and macrophages in the peribronchial space, as well as collagen fiber accumulation, epithelial thickness, and mucus accumulation. Furthermore, APT lowered the expression of the B1 receptor and ACE-2 in lung tissue and reduced bradykinin levels in the lung tissue homogenate compared to the HDM group. It also improved airway resistance, tissue resistance, and tissue damping. On a systemic level, APT reduced total leukocytes, eosinophils, neutrophils, basophils, lymphocytes, and monocytes in the blood, as well as plasma levels of IL-1β, IL-4, IL-5, IL-17, TNF-α, and IL-33, while elevating the levels of IL-10 and IL-1RA. These findings indicate that APT inhibits the severe asthma phenotype by targeting kinin signaling.

Exploring alveolar recruitability using positive end-expiratory pressure in mice overexpressing TGF-β1: a structure–function analysis

Pre-injured lungs are prone to injury progression in response to mechanical ventilation. Heterogeneous ventilation due to (micro)atelectases imparts injurious strains on open alveoli (known as volutrauma). Hence, recruitment of (micro)atelectases by positive end-expiratory pressure (PEEP) is necessary to interrupt this vicious circle of injury but needs to be balanced against acinar overdistension. In this study, the lung-protective potential of alveolar recruitment was investigated and balanced against overdistension in pre-injured lungs. Mice, treated with empty vector (AdCl) or adenoviral active TGF-β1 (AdTGF-β1) were subjected to lung mechanical measurements during descending PEEP ventilation from 12 to 0 cmH2O. At each PEEP level, recruitability tests consisting of two recruitment maneuvers followed by repetitive forced oscillation perturbations to determine tissue elastance (H) and damping (G) were performed. Finally, lungs were fixed by vascular perfusion at end-expiratory airway opening pressures (Pao) of 20, 10, 5 and 2 cmH2O after a recruitment maneuver, and processed for design-based stereology to quantify derecruitment and distension. H and G were significantly elevated in AdTGF-β1 compared to AdCl across PEEP levels. H was minimized at PEEP = 5–8 cmH2O and increased at lower and higher PEEP in both groups. These findings correlated with increasing septal wall folding (= derecruitment) and reduced density of alveolar number and surface area (= distension), respectively. In AdTGF-β1 exposed mice, 27% of alveoli remained derecruited at Pao = 20 cmH2O. A further decrease in Pao down to 2 cmH2O showed derecruitment of an additional 1.1 million alveoli (48%), which was linked with an increase in alveolar size heterogeneity at Pao = 2–5 cmH2O. In AdCl, decreased Pao resulted in septal folding with virtually no alveolar collapse. In essence, in healthy mice alveoli do not derecruit at low PEEP ventilation. The potential of alveolar recruitability in AdTGF-β1 exposed mice is high. H is optimized at PEEP 5–8 cmH2O. Lower PEEP folds and larger PEEP stretches septa which results in higher H and is more pronounced in AdTGF-β1 than in AdCl. The increased alveolar size heterogeneity at Pao = 5 cmH2O argues for the use of PEEP = 8 cmH2O for lung protective mechanical ventilation in this animal model.

Kirchhoff's law-based velocity-controlled motion models to predict real-time cutting forces in minimally invasive surgeries

A theoretical framework, united by a “system effect” is formulated to model the cutting/haptic force evolution at the cutting edge of a surgical cutting instrument during its penetration into soft biological tissue in minimally invasive surgery. Other cutting process responses, including tissue fracture force, friction force, and damping, are predicted by the model as well. The model is based on a velocity-controlled formulation of the corresponding equations of motion, derived for a surgical cutting instrument and tissue based on Kirchhoff's fundamental energy conservation law. It provides nearly zero residues (absolute errors) in the equations of motion balances. In addition, concurrent closing relationships for the fracture force, friction coefficient, friction force, process damping, strain rate function (a constitutive tissue model), and their implementation within the proposed theoretical framework are established. The advantage of the method is its ability to make precise real-time predictions of the aperiodic fluctuating evolutions of the cutting forces and the other process responses. It allows for the robust modeling of the interactions between a medical instrument and a nonlinear viscoelastic tissue under any physically feasible working conditions. The cutting process model was partially qualitatively verified through numerical simulations and by comparing the computed cutting forces with experimentally measured values during robotic uniaxial biopsy needle constant velocity insertion into artificial gel tissue, obtained from previous experimental research. The comparison has shown a qualitatively similar adequate trend in the evolution of the experimentally measured and numerically predicted cutting forces during insertion of the needle.

Mucopolysaccharidosis (MPS IIIA) mice have increased lung compliance and airway resistance, decreased diaphragm strength, and no change in alveolar structure.

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterized by neurological and skeletal pathologies caused by reduced activity of the lysosomal hydrolase, sulfamidase, and the subsequent primary accumulation of undegraded heparan sulfate (HS). Respiratory pathology is considered secondary in MPS IIIA and the mechanisms are not well understood. Changes in the amount, metabolism, and function of pulmonary surfactant, the substance that regulates alveolar interfacial surface tension and modulates lung compliance and elastance, have been reported in MPS IIIA mice. Here we investigated changes in lung function in 20-wk-old control and MPS IIIA mice with a closed and open thoracic cage, diaphragm contractile properties, and potential parenchymal remodeling. MPS IIIA mice had increased compliance and airway resistance and reduced tissue damping and elastance compared with control mice. The chest wall impacted lung function as observed by an increase in airway resistance and a decrease in peripheral energy dissipation in the open compared with the closed thoracic cage state in MPS IIIA mice. Diaphragm contractile forces showed a decrease in peak twitch force, maximum specific force, and the force-frequency relationship but no change in muscle fiber cross-sectional area in MPS IIIA mice compared with control mice. Design-based stereology did not reveal any parenchymal remodeling or destruction of alveolar septa in the MPS IIIA mouse lung. In conclusion, the increased storage of HS which leads to biochemical and biophysical changes in pulmonary surfactant also affects lung and diaphragm function, but has no impact on lung or diaphragm structure at this stage of the disease.NEW & NOTEWORTHY Heparan sulfate storage in the lungs of mucopolysaccharidosis type IIIA (MPS IIIA) mice leads to changes in lung function consistent with those of an obstructive lung disease and includes an increase in lung compliance and airway resistance and a decrease in tissue elastance. In addition, diaphragm muscle contractile strength is reduced, potentially further contributing to lung function impairment. However, no changes in parenchymal lung structure were observed in mice at 20 wk of age.

Antenatal Administration of Extracellular Vesicles Derived From Amniotic Fluid Stem Cells Improves Lung Function in Neonatal Rats With Congenital Diaphragmatic Hernia

BackgroundThe severity of pulmonary hypoplasia is a main determinant of outcome for babies with congenital diaphragmatic hernia (CDH). Antenatal administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) has been shown to rescue morphological features of lung development in the rat nitrofen model of CDH. Herein, we evaluated whether AFSC-EV administration to fetal rats with CDH is associated with neonatal improvement in lung function. MethodsAFSC-EVs were isolated by ultracentrifugation and characterized by size, morphology, and canonical marker expression. At embryonic (E) day 9.5, dams were gavaged with olive oil (control) or nitrofen to induce CDH. At E18.5, fetuses received an intra-amniotic injection of either saline or AFSC-EVs. At E21.5, rats were delivered and subjected to a tracheostomy for mechanical ventilation (flexiVent system). Groups were compared for lung compliance, resistance, Newtonian resistance, tissue damping and elastance. Lungs were evaluated for branching morphogenesis and collagen quantification. ResultsCompared to healthy control, saline-treated pups with CDH had fewer airspaces, more collagen deposition, and functionally exhibited reduced compliance and increased airway resistance, elastance, and tissue damping. Conversely, AFSC-EV administration resulted in improvement of lung mechanics (compliance, resistance, tissue damping, elastance) as well as lung branching morphogenesis and collagen deposition. ConclusionsOur studies show that the rat nitrofen model reproduces lung function impairment similar to that of human babies with CDH. Antenatal administration of AFSC-EVs improves lung morphology and function in neonatal rats with CDH. Level of EvidenceN/A (animal and laboratory study).

Changes in lung mechanics and ventilation-perfusion match: comparison of pulmonary air- and thromboembolism in rats

BackgroundPulmonary air embolism (AE) and thromboembolism lead to severe ventilation-perfusion defects. The spatial distribution of pulmonary perfusion dysfunctions differs substantially in the two pulmonary embolism pathologies, and the effects on respiratory mechanics, gas exchange, and ventilation-perfusion match have not been compared within a study. Therefore, we compared changes in indices reflecting airway and respiratory tissue mechanics, gas exchange, and capnography when pulmonary embolism was induced by venous injection of air as a model of gas embolism or by clamping the main pulmonary artery to mimic severe thromboembolism.MethodsAnesthetized and mechanically ventilated rats (n = 9) were measured under baseline conditions after inducing pulmonary AE by injecting 0.1 mL air into the femoral vein and after occluding the left pulmonary artery (LPAO). Changes in mechanical parameters were assessed by forced oscillations to measure airway resistance, lung tissue damping, and elastance. The arterial partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2) were determined by blood gas analyses. Gas exchange indices were also assessed by measuring end-tidal CO2 concentration (ETCO2), shape factors, and dead space parameters by volumetric capnography.ResultsIn the presence of a uniform decrease in ETCO2 in the two embolism models, marked elevations in the bronchial tone and compromised lung tissue mechanics were noted after LPAO, whereas AE did not affect lung mechanics. Conversely, only AE deteriorated PaO2, and PaCO2, while LPAO did not affect these outcomes. Neither AE nor LPAO caused changes in the anatomical or physiological dead space, while both embolism models resulted in elevated alveolar dead space indices incorporating intrapulmonary shunting.ConclusionsOur findings indicate that severe focal hypocapnia following LPAO triggers bronchoconstriction redirecting airflow to well-perfused lung areas, thereby maintaining normal oxygenation, and the CO2 elimination ability of the lungs. However, hypocapnia in diffuse pulmonary perfusion after AE may not reach the threshold level to induce lung mechanical changes; thus, the compensatory mechanisms to match ventilation to perfusion are activated less effectively.

Adjuvant effect of inhaled particulate matter containing free radicals following house-dust mite induction of asthma in mice

Introduction Exposures to particulate matter (PM) from combustion sources can exacerbate preexisting asthma. However, the cellular and molecular mechanisms by which PM promotes the exacerbation of asthma remain elusive. We used a house dust mite (HDM)-induced mouse model of asthma to test the hypothesis that inhaled DCB230, which are PM containing environmentally persistent free radicals (EPFRs), will aggravate asthmatic responses. Methods Groups of 8–10-week-old C57BL/6 male mice were exposed to either air or DCB230 aerosols at a concentration of 1.5 mg/m3 4 h/day for 10 days with or without prior HDM-induction of asthma. Results Aerosolized DCB230 particles formed small aggregates (30–150 nm). Mice exposed to DCB230 alone showed significantly reduced lung tidal volume, overexpression of the Muc5ac gene, and dysregulation of 4 inflammation related genes, Ccl11, Ccl24, Il-10, and Tpsb2. This suggests DCB230 particles interacted with the lung epithelium inducing mucous hypersecretion and restricting lung volume. In addition to reduced lung tidal volume, compared to respective controls, the HDM + DCB230-exposed group exhibited significantly increased lung tissue damping and up-regulated expression of Muc5ac, indicating that in this model, mucous hypersecretion may be central to pulmonary dysfunction. This group also showed augmented lung eosinophilic inflammation accompanied by an up-regulation of 36 asthma related genes. Twelve of these genes are part of IL-17 signaling, suggesting that this pathway is critical for DCB230 induced toxicity and adjuvant effects in lungs previously exposed to HDM. Conclusion Our data indicate that inhaled DCB230 can act as an adjuvant, exacerbating asthma through IL-17-mediated responses in a HDM mouse model.

Lung mechanics showing sex-based differences and circadian time-of-day response to bleomycin-induced lung injury in mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that impairs lung mechanical properties due to dysregulated extracellular matrix remodeling. Lung function assessment is an important physiological endpoint in the mouse model of pulmonary fibrosis (PF) that has gained a broader scientific acceptance in the field. IPF pathophysiology shows sex-based differences, disproportionately affecting more men compared to women. Prior reports suggest that the circadian clock is perturbed during the pathogenesis of PF. We have comprehensively assessed the sex-based differences and time-of-day response (at Zeitgeber time: ZT0/6:00 a.m. or ZT12/6 p.m.) in lung mechanics among sham (control) versus bleomycin (BLM)-challenged female and male (C57BL/6: WT) mice using Flexi-vent. BLM challenge altered lung function significantly in males in both total lung (reduced dynamic compliance, and increased resistance and elastance) as well as lung tissue-specific parameters (increased tissue elastance and tissue damping) but less pronounced in females. BLM-challenged mice showed a time-of-day response in lung function at ZT0 versus ZT12, which was pronounced in the ZT0 BLM group. Overall, these findings provide a comprehensive analysis of altered lung function in female and male mice and the time-of-day difference in lung function parameters following BLM-induced lung fibrosis.

Postnatal growth restriction impairs rat lung structure and function.

The negative impact of nutritional deficits in the development of bronchopulmonary dysplasia is well recognized, yet mechanisms by which nutrition alters lung outcomes and nutritional strategies that optimize development and protect the lung remain elusive. Here, we use a rat model to assess the isolated effects of postnatal nutrition on lung structural development without concomitant lung injury. We hypothesize that postnatal growth restriction (PGR) impairs lung structure and function, critical mediators of lung development, and fatty acid profiles at postnatal day 21 in the rat. Rat pups were cross-fostered at birth to rat dams with litter sizes of 8 (control) or 16 (PGR). Lung structure and function, as well as serum and lung tissue fatty acids, and lung molecular mediators of development, were measured. Male and female PGR rat pups had thicker airspace walls, decreased lung compliance, and increased tissue damping. Male rats also had increased lung elastance, increased lung elastin protein abundance, and lysol oxidase expression, and increased elastic fiber deposition. Female rat lungs had increased conducting airway resistance and reduced levels of docosahexaenoic acid in lung tissue. We conclude that PGR impairs lung structure and function in both male and female rats, with sex-divergent changes in lung molecular mediators of development.

Characterization of middle ear soft tissue damping and its role in sound transmission

Damping plays an important role in the middle ear (ME) sound transmission system. However, how to mechanically characterize the damping of ME soft tissues and the role of damping in ME sound transmission have not yet reached a consensus. In this paper, a finite element (FE) model of the partial external and ME of the human ear, considering both Rayleigh damping and viscoelastic damping for different soft tissues, is developed to quantitatively investigate the damping in soft tissues effects on the wide-frequency response of the ME sound transmission system. The model-derived results can capture the high-frequency (above 2kHz) fluctuations and obtain the 0.9kHz resonant frequency (RF) of the stapes velocity transfer function (SVTF) response. The results show that the damping of pars tensa (PT), stapedial annular ligament (SAL) and incudostapedial joints (ISJ) can help smooth the broadband response of the umbo and stapes footplate (SFP). It is found that, between 1 and 8kHz, the damping of the PT increases the magnitude and phase delay of the SVTF above 2kHz while the damping of the ISJ can avoid excessive phase delay of the SVTF, which is important in maintaining the synchronization in high-frequency vibration but has not been revealed before. Below 1kHz, the damping of the SAL plays a more important role, and it can decrease the magnitude but increases the phase delay of the SVTF. This study has implications for a better understanding of the mechanism of ME sound transmission.

Distinct type I collagen alterations cause intrinsic lung and respiratory defects of variable severity in mouse models of osteogenesis imperfecta.

Type I collagen alterations cause osteogenesis imperfecta (OI), a connective tissue disorder characterized by severe bone fragility. Patients with OI can suffer from significant pulmonary manifestations including severe respiratory distress in the neonatal period and a progressive decline in respiratory function in adulthood. We and others have shown intrinsic lung defects in some mouse models of OI. In this large study, we performed histological, histomorphometric, microcomputed tomography and invasive studies on oim/+, Col1a2+/G610C , CrtapKO and oim/oim mice, mimicking mild to moderate to severe OI, with the overall goal of determining the extent of their pulmonary and respiratory mechanics defects and whether these defects correlate with the skeletal disease severity and affect each sex equally. Although with variable severity, OI lung histology consistently showed alveolar simplification with enlarged acinar airspace and reduced alveolar surface. Numerous respiratory mechanics parameters, including respiratory system resistance and elastance, tissue damping, inspiratory capacity, total lung capacity, and others, were significantly and similarly impacted in CrtapKO and oim/oim but not in oim/+ or Col1a2+/G610C compared to control mice. Our data indicate that the impact of type I collagen alterations and OI on lung morphology and function positively correlate with the severity of the extracellular matrix deficiency. Moreover, the respiratory defects were more pronounced in male compared to female mice. It will be important to determine whether our observations in mice translate to OI patients and to dissect the respective contribution of intrinsic lung defects vs. extrinsic skeletal defects to impaired lung function in OI. KEY POINTS: Different type I collagen alterations in mouse models of osteogenesis imperfecta (OI) cause similar abnormal lung histology, with alveolar simplification and reduced alveolar surface, reminiscent of emphysema. Several respiratory mechanics parameters are altered in mouse models of OI. The impact of type I collagen alterations and OI on lung morphology and function positively correlate with the severity of the extracellular matrix deficiency. Respiratory defects were more pronounced in male compared to female mice. It will be important to determine whether our observations in mice translate to OI patients and to dissect the respective contribution of intrinsic lung defects vs. extrinsic skeletal defects to impaired lung function in OI.

Flow-controlled ventilation maintains gas exchange and lung aeration in a pediatric model of healthy and injured lungs: A randomized cross-over experimental study.

Flow-controlled ventilation (FCV) is characterized by a constant flow to generate active inspiration and expiration. While the benefit of FCV on gas exchange has been demonstrated in preclinical and clinical studies with adults, the value of this modality for a pediatric population remains unknown. Thus, we aimed at observing the effects of FCV as compared to pressure-regulated volume control (PRVC) ventilation on lung mechanics, gas exchange and lung aeration before and after surfactant depletion in a pediatric model. Ten anesthetized piglets (10.4 ± 0.2 kg) were randomly assigned to start 1-h ventilation with FCV or PRVC before switching the ventilation modes for another hour. This sequence was repeated after inducing lung injury by bronchoalveolar lavage and injurious ventilation. The primary outcome was respiratory tissue elastance. Secondary outcomes included oxygenation index (PaO2/FiO2), PaCO2, intrapulmonary shunt (Qs/Qt), airway resistance, respiratory tissue damping, end-expiratory lung volume, lung clearance index and lung aeration by chest electrical impedance tomography. Measurements were performed at the end of each protocol stage. Ventilation modality had no effect on any respiratory mechanical parameter. Adequate gas exchange was provided by FCV, similar to PRVC, with sufficient CO2 elimination both in healthy and surfactant-depleted lungs (39.46 ± 7.2 mmHg and 46.2 ± 11.4 mmHg for FCV; 36.0 ± 4.1 and 39.5 ± 4.9 mmHg, for PRVC, respectively). Somewhat lower PaO2/FiO2 and higher Qs/Qt were observed in healthy and surfactant depleted lungs during FCV compared to PRVC (p < 0.05, for all). Compared to PRVC, lung aeration was significantly elevated, particularly in the ventral dependent zones during FCV (p < 0.05), but this difference was not evidenced in injured lungs. Somewhat lower oxygenation and higher shunt ratio was observed during FCV, nevertheless lung aeration improved and adequate gas exchange was ensured. Therefore, in the absence of major differences in respiratory mechanics and lung volumes, FCV may be considered as an alternative in ventilation therapy of pediatric patients with healthy and injured lungs.

217.9: Activation of Angiotensin-converting Enzyme 2 (ACE2) Modulates Lung Mechanics in a Brain Death Model and Attenuates Pulmonary Edema After Rat Lung Transplantation

Introduction: Lung transplantation is a therapeutic option established worldwide for end-stage lung diseases. However, as most donors evolve with brain death (BD), the donated lung may have been compromised. In view of the participation of the renin angiotensin system (RAS) and the use of angiotensin-converting enzyme 2 (ACE2) activators in several pathological processes, it was investigated how diminazene aceturate (DIZE), an ACE2 activator, modulates hemodynamic and blood gas changes, as well as lung function following transplantation in rats. Method: Male Lewis rats were randomly distributed in the following experimental groups: intact, brain death (BD) and BD + diminazene aceturate (BD+DIZE). The last two groups were submitted to brain death process, treated with saline (0.9%) or DIZE (15 mg/kg-1) and kept on mechanical ventilation for 6 hours (donor). After this period, cardiopulmonary block extraction and left unilateral transplantation (LTx) with independent graft ventilation was performed, followed by 2h reperfusion. Hemodynamics (mean arterial pressure - MAP), blood gas analysis, lung mechanics, and pulmonary edema were evaluated. Results are presented as S.E.M. (ANOVA; Tukey; p<0.05). (Approval # CEUA/FMUSP - 1630/2021). Results: The administration of DIZE, 3 hours after BD, prevented the increase in the MAP (210’ after BD-induction: BD – 110 ± 12 mmHg; n=5; BD+DIZE – 40 ± 12 mmHg; n=4/ p=0.0001) without associated blood gas and metabolic alterations. Changes in lung mechanics, such as decreased tissue damping (G) and histeresivity (n) followed by increased respiratory system compliance (Crs), were associated with BD+DIZE (G - 0,25 ± 0,03 cmH2O/mL; Crs - 0,71 ± 0,03 mL/cmH2O; n – 0,14 ± 0,008) compared to intact (G - 0,40 ± 0,02 cmH2O/mL, p = 0.01; Crs – 0,64 ± 0,01 mL/cmH2O, p = 0.02; n - 0,26 ± 0,01, p = 0.004) and BD groups (G – 0,41± 0,05 cmH2O/mL, p = 0.02; Crs – 0,61 ± 0,01 mL/cmH2O, p = 0.01; n - 0,25 ± 0,008, p = 0.01) (Figure 1). After LTx, MAP, gas exchange function, and lung mechanical parameters, were not different among the 3 groups (p>0.05). On the other hand, the DIZE treatment was capable to reduce wet-to-dry weight ratio (INTACT – 1,87 ± 0,13; BD – 2,59 ± 0,14; BD+DIZE – 1,93 ± 0,15/ p=0.01) (Figure 2). Conclusion: Our data suggests that activation of ACE2 is able to modulate donor hemodynamic and lung mechanical parameters, probably reducing tissue resistance and improving airflow in the peripheral airways, converging to a decrease in ventilation heterogeneity in the sudden onset model of brain death. Our data also indicates that DIZE attenuates pulmonary edema on grafts from BD donors after lung transplantation. Keywords: Diminazene aceturate. Renin-angiotensin system. Brain death. Lung mechanics. Lung transplantation.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico.

Abstract P2027: Short-term Ambient Particulate Exposure And Social Defeat Cause Reductions In Heart And Pulmonary Function

Deployed service members and first responders are exposed to environments containing high levels of air pollution and psychological distress. Thus, we sought to investigate the interaction of these factors and whether the combination of PM and stress exposures exacerbate cardiac and lung dysfunction in a mouse model. Male C57BL/6 mice were exposed to filtered air (FA) or PM (≤2.5 μm) at concentrations of 137.7 ± 70.76 μg/m 3 (daily mean ± standard deviation) for five days/week for three weeks. A subset of each group was stressed via exposure to a retired male aggressor for two hours/day, 3-5 nights/week (FA-stress and PM-stress groups). Echocardiography revealed no changes in left ventricle (LV) systolic or diastolic indices, but a significant reduction in right ventricle (RV) stroke volume and cardiac output in the PM, FA-stress and PM-stress groups compared to the FA group (P&lt;0.05 via two-way ANOVA). Pressure-volume loops displayed significantly reduced end-systolic elastance for the PM and PM-stress groups, but not for the FA-stress group when compared to FA controls, indicating reduced contractility (Ees, P&lt;0.05 via two-way ANOVA). Data on respiratory mechanics was acquired using the flexiVent FX2 (SCIREQ Inc.) system. PM exposure caused an increase in lung resistance (Rrs), central airway resistance (Rn), and tissue damping (G) compared to FA (P&lt;0.05 via two-way ANOVA at individual doses of methacholine). Also, the FA-stress and PM-stress groups experienced increased lung elastance (Ers), Rrs, Rn, and G compared to FA mice (P&lt;0.05 via two-way ANOVA at individual doses). Tail-vein blood pressure measurements showed an increased diastolic pressure induced by PM exposure, but not by stress (P&lt;0.05 via two-way ANOVA). RT-qPCR data revealed a significant decrease in RV NPPB gene expression and an increased RV Col1A1 expression for PM-exposed mice compared to FA (via two-way ANOVA). Additionally, RT-qPCR demonstrated that PM decreased the expression of many inflammatory markers (TNF-α, CCL2, IL6, and IL-1β) in the LV and RV relative to FA (P&lt;0.05 via two-way ANOVA). Differences in NPPB and Col1A1 expression suggest increased fibrosis in the RV. Calculating capillary to myocyte ratio from a wheat germ agglutinin stain, there was an increase in capillary density of PM-exposed mice compared to FA (P&lt;0.05 via t-test). Taken together, short-term exposure to PM with/without psychological distress impairs respiratory mechanics and RV function. Molecular analyses indicated changes in inflammation in both the LV and RV between the FA and PM groups, with additional changes in RV fibrosis. These results suggest that even short-term exposure to PM, including in occupational settings such as military burn pits, can cause notable cardiopulmonary dysfunction. This work was supported by the Department of Veterans Affairs Airborne Hazards and Burn Pits Center of Excellence

Exogenous leptin enhances markers of airway fibrosis in a mouse model of chronic allergic airways disease

BackgroundAsthma patients with comorbid obesity exhibit increased disease severity, in part, due to airway remodeling, which is also observed in mouse models of asthma and obesity. A mediator of remodeling that is increased in obesity is leptin. We hypothesized that in a mouse model of allergic airways disease, mice receiving exogenous leptin would display increased airway inflammation and fibrosis.MethodsFive-week-old male and female C57BL/6J mice were challenged with intranasal house dust mite (HDM) allergen or saline 5 days per week for 6 weeks (n = 6–9 per sex, per group). Following each HDM exposure, mice received subcutaneous recombinant human leptin or saline. At 48 h after the final HDM challenge, lung mechanics were evaluated and the mice were sacrificed. Bronchoalveolar lavage was performed and differential cell counts were determined. Lung tissue was stained with Masson’s trichrome, periodic acid-Schiff, and hematoxylin and eosin stains. Mouse lung fibroblasts were cultured, and whole lung mRNA was isolated.ResultsLeptin did not affect mouse body weight, but HDM+leptin increased baseline blood glucose. In mixed-sex groups, leptin increased mouse lung fibroblast invasiveness and increased lung Col1a1 mRNA expression. Total lung resistance and tissue damping were increased with HDM+leptin treatment, but not leptin or HDM alone. Female mice exhibited enhanced airway responsiveness to methacholine with HDM+leptin treatment, while leptin alone decreased total respiratory system resistance in male mice.ConclusionsIn HDM-induced allergic airways disease, administration of exogenous leptin to mice enhanced lung resistance and increased markers of fibrosis, with differing effects between males and females.

Effect of prolonged lipopolysaccharide exposure on lung function and health

RationalePatients with Chronic Obstructive Pulmonary Disease often have persistent inflammation of the airways that lead to symptoms of chronic bronchitis and airway obstruction. While it is known that long term exposure to cigarette smoke can cause persistent airways inflammation, it is not clear if this is a direct result of tobacco exposure or if it is a result of chronic inflammation. We hypothesized that a recurrent and prolonged inflammatory insult would result in alterations in lung mechanics characterized by airway obstruction.Methods. To test the independent effects of recurrent and persistent lung inflammation on lung mechanics, C57Bl/6 8 week old male mice were treated weekly with PBS or lipopolysaccharide (LPS) via direct intratracheal injection. Monthly, we evaluated lung function analysis including: airway resistance and compliance, tissue damping, pressure volume loops, and forced expiratory volume using the flexiVent system.Results. Airway resistance at month 1 was 0.5543±0.017 PBS vs 0.552±0.009 cmH2O.s/mL LPS (p=0.999), month 2 0.5177±0.015 PBS vs 0.576±0.017 LPS (p=0.999), month 3 0.461± 0.03 PBS vs 0.47±0.038 cmH2O.s/mL LPS (p=0.999), month 5 0.43±0.015 PBS vs 0.468±0.026 cmH2O.s/mL LPS (p=0.999), month 6 PBS 0.408± 0.06 vs LPS 0.6465±0.18 cmH2O.s/mL (p=0.999), and month 7 PBS 0.384 ± 0.023 vs LPS 0.3498 ± 0.015 cmH2O.s/mL (p=0.999). Forced expiratory volume 0.1 (FEV 0.1) at month 1 was 0.4±0.08 PBS vs 0.803±0.107 mL LPS (p=0.999), month 2 0.2115±0.01 PBS vs 0.184±0.03 mL LPS (p=0.999), month 3 1.071±0.09 PBS vs 1.098±0.06 mL LPS (p=0.999), month 5 0.853±0.243 vs 0.756±0.21 mL LPS (p=0.999), month 6 2.671±1.06 PBS vs 1.669±0.87 mL LPS (p=0.999), month 7 0.9049±0.27 PBS vs 1.127±0.152 mL LPS (p=0.999).ConclusionIt is well known that chronic smoking can have a significant impact of lung function. Through this project we aimed to discover whether chronic inflammation alone would alter lung function. Interestingly even after weekly direct intratracheal injections of LPS there were no significant changes in lung function compared to PBS treated mice. We have shown here that chronic inflammation alone was not sufficient to alter lung function in mice, and that the decrease in lung function seen in chronic smokers is attributable to numerous factors and not inflammation alone.

Loss of Club Cell Creb Mitigates IL‐1B–mediated Muco‐obstructive Phenotypes in Male Murine Airways

Goblet cell metaplasia and/or mucus hypersecretion are hallmark features of multiple lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and pulmonary fibrosis. Interleukin 1β (IL‐1B) is a proinflammatory mediator that increases expression of the major gel forming mucins in the airways, Muc5b and Muc5ac, in part through activation of the cAMP response element binding protein (Creb) transcription factor. IL‐1B also induces Spdef, a key regulator of goblet cell differentiation from airway club cells. Here we tested the hypothesis that elimination of murine club cell Creb mitigates muco‐obstructive phenotypes induced by IL‐1B. Mice with floxed Creb1 were bred to Scgb1atm1(cre/ERT)Blh to generate mice with conditional loss of Creb1 from club cells. Wild‐type and transgenic male mice (7‐9/group) littermates received intranasal IL‐1B (0.5 ng/inhalation) or saline (control) for four consecutive days, with tamoxifen administration occurring on days 1 and 3 to induce Cre recombinase expression. Periodic acid–Schiff staining in lung samples showed that IL‐1B increased the number of goblet cells in central airways compared to saline controls. This effect was blunted by conditional loss of Creb1. Similarly, flexiVent measurements demonstrated that IL‐1B increased airway resistance (R), Newtonian resistance (Rn), tissue elastance (ERS), tissue damping (G) and tissue elasticity (H). Loss of club cell Creb1 mitigated all effects of IL‐1B on airway mechanics. Additionally, though the percentage of granulocytes in the bronchoalveolar lung lavage fluid was not increased by IL‐1B, loss of club cell Creb1 decreased the percentage of granulocytes in the bronchoalveolar lung lavage fluid independent of treatment (i.e., saline or IL‐1B). These findings suggest that the muco‐obstructive effects of IL‐1B are dependent upon club cell Creb1 and provide novel insight for therapeutic strategies.

Kisspeptin Reduces Airway Hyperreactivity and Remodeling in Asthmatic Ovariectomized Mice

RationaleEpidemiological data show changes in asthma occurrence with age and sex. After puberty, increased severity in women shows sex‐skewed occurrence of asthma in females vs. males. This implies the importance of female sex‐steroids, particularly estrogen in asthma pathophysiology. Greater asthma in women may represent detrimental mechanisms upstream to or independent of estrogen. Moreover, data from CNS studies show kisspeptin (Kp) signaling is modulated by estrogen. Our recent in vitro studies show Kp via its receptor, KISS1R regulates airway smooth muscle (ASM) cell proliferation. However, the relevance of endogenous estrogen on Kp/KISS1R signaling and their effect on regulating airway hyperresponsiveness (AHR) and remodeling is not yet explored. Accordingly, in this present study, we utilized intact female vs. ovariectomized (OVX) mice to isolate estrogen influence from the Kp effect in regulating AHR and remodeling in a murine model of allergic asthma.MethodsWild Type C57BL/6J intact female and OVX mice were procured from Jackson Laboratory, USA, and challenged intranasally with a mixed‐allergen (MA) for 28 days. On alternate days, animals from the treatment group received intranasal Kp‐10 (1.2mg/kg), vehicle group received PBS alone. On day 29, mice were anesthetized and subjected to SCIREQ flexiVent to measure lung mechanics like airway resistance (Rrs), elastance (Ers), compliance (Crs), tissue damping (G), and tissue‐elasticity (H). Following this, bronchoalveolar lavage (BAL) was performed for differential leukocyte count (DLC) and lung tissues were processed for histology using hematoxylin and eosin (H&amp;E) and trichrome staining. Additionally, laser‐capture microdissection (LCM) assisted qRT‐PCR analysis in ASM cells was performed for genes relevant to airway remodeling.ResultsMA‐challenged intact female and OVX mice showed a significant increase in Rrs, Ers, G, H, and reduced Crs compared to PBS, more pronounced effects were observed in intact females. Kp‐10 treatment significantly reversed the MA‐induced changes with a decrease in Rrs, Ers, G, H, and increased Crs. Interestingly, in OVX mice we observed significant effects of Kp‐10 to improve the lung mechanics compared to intact females. Notably, Kp‐10 alone exposed group did not show any changes in lung mechanics compared to PBS. DLC in BAL showed Kp‐10 treatment significantly reduced MA‐induced inflammatory cell infiltration. Futhermore, histology studies showed Kp‐10 treatment significantly reduced MA‐induced smooth muscle mass and collagen deposition in airways.ConclusionOverall, this study shows the modulatory role of estrogen in regulating kisspeptin signaling, thereby AHR and remodeling. Thus, providing the relevance to higher asthma incidence and severity in women compared to men.

The Heat Shock Protein 90 Inhibitor, AT13387, Protects the Alveolo-Capillary Barrier and Prevents HCl-Induced Chronic Lung Injury and Pulmonary Fibrosis.

Hydrochloric acid (HCl) exposure causes asthma-like conditions, reactive airways dysfunction syndrome, and pulmonary fibrosis. Heat Shock Protein 90 (HSP90) is a molecular chaperone that regulates multiple cellular processes. HSP90 inhibitors are undergoing clinical trials for cancer and are also being studied in various pre-clinical settings for their anti-inflammatory and anti-fibrotic effects. Here we investigated the ability of the heat shock protein 90 (HSP90) inhibitor AT13387 to prevent chronic lung injury induced by exposure to HCl in vivo and its protective role in the endothelial barrier in vitro. We instilled C57Bl/6J mice with 0.1N HCl (2 µL/g body weight, intratracheally) and after 24 h began treatment with vehicle or AT13387 (10 or 15 mg/kg, SC), administered 3×/week; we analyzed histological, functional, and molecular markers 30 days after HCl. In addition, we monitored transendothelial electrical resistance (TER) and protein expression in a monolayer of human lung microvascular endothelial cells (HLMVEC) exposed to HCl (0.02 N) and treated with vehicle or AT13387 (2 µM). HCl provoked persistent alveolar inflammation; activation of profibrotic pathways (MAPK/ERK, HSP90); increased deposition of collagen, fibronectin and elastin; histological evidence of fibrosis; and a decline in lung function reflected in a downward shift in pressure–volume curves, increased respiratory system resistance (Rrs), elastance (Ers), tissue damping (G), and hyperresponsiveness to methacholine. Treatment with 15 mg/kg AT13387reduced alveolar inflammation, fibrosis, and NLRP3 staining; blocked activation of ERK and HSP90; and attenuated the deposition of collagen and the development of chronic lung injury and airway hyperreactivity. In vitro, AT13387 prevented HCl-induced loss of barrier function and AKT, ERK, and ROCK1 activation, and restored HSP70 and cofilin expression. The HSP90 inhibitor, AT13387, represents a promising drug candidate for chronic lung injury that can be administered subcutaneously in the field, and at low, non-toxic doses.