KF4 anti-CELA1 Antibody and Purified α1-Antitrypsin Have Similar but Not Additive Efficacy in Preventing Emphysema in Murine α1-Antitrypsin Deficiency.

Alpha-1 antitrypsin (AAT) deficiency is the most common genetic cause of emphysema. Chymotrypsin-like Elastase 1 (CELA1) is a serine protease neutralized by AAT and is important in emphysema progression. Cela1-deficiency is protective in a murine models of AAT-deficient emphysema. KF4 anti-CELA1 antibody prevented emphysema in PPE and cigarette smoke models in wild type mice. We evaluated potential toxicities of KF4 and its ability to prevent emphysema in AAT deficiency. We found Cela1 protein expression in mouse lung, pancreas, small intestine, and spleen. In toxicity studies, mice treated with KF4 25 mg/kg weekly for four weeks showed an elevation in blood urea nitrogen and slower weight gain compared to lower doses or equivalent dose IgG. In histologic grading of tissue injury of the lung, kidney, liver, and heart, there was some evidence of liver injury with KF4 25 mg/kg, but in all tissues, injury was less than in control mice subjected to cecal ligation and puncture. In efficacy studies, KF4 doses as low as 0.5 mg/kg reduced the lung elastase activity of AAT-/- mice treated with 0.2 units of PPE. In this injury model, AAT-/- mice treated with KF4 1 mg/kg weekly, human purified AAT 60 mg/kg weekly, and combined KF4 and AAT treatment had less emphysema than mice treated with IgG 1 mg/kg weekly. However, the efficacy of KF4, AAT, or KF4 & AAT was similar. While KF4 might be an alternative to AAT replacement, combined KF4 and AAT replacement does not confer additional benefit.

Anti-CELA1 antibody KF4 prevents emphysema by inhibiting stretch-mediated remodeling.

There are no therapies to prevent emphysema progression. Chymotrypsin-like elastase 1 (CELA1) is a serine protease that binds and cleaves lung elastin in a stretch-dependent manner and is required for emphysema in a murine antisense oligonucleotide model of α-1 antitrypsin (AAT) deficiency. This study tested whether CELA1 is important in strain-mediated lung matrix destruction in non-AAT-deficient emphysema and the efficacy of CELA1 neutralization. Airspace simplification was quantified after administration of tracheal porcine pancreatic elastase (PPE), after 8 months of cigarette smoke (CS) exposure, and in aging. In all 3 models, Cela1-/- mice had less emphysema and preserved lung elastin despite increased lung immune cells. A CELA1-neutralizing antibody was developed (KF4), and it inhibited stretch-inducible lung elastase in ex vivo mouse and human lung and immunoprecipitated CELA1 from human lung. In mice, systemically administered KF4 penetrated lung tissue in a dose-dependent manner and 5 mg/kg weekly prevented emphysema in the PPE model with both pre- and postinjury initiation and in the CS model. KF4 did not increase lung immune cells. CELA1-mediated lung matrix remodeling in response to strain is an important contributor to postnatal airspace simplification, and we believe that KF4 could be developed as a lung matrix-stabilizing therapy in emphysema.

A titratable murine model of progressive emphysema using tracheal porcine pancreatic elastase.

Progressive emphysema often leads to end-stage lung disease. Most mouse models of emphysema are typically modest (i.e. cigarette smoke exposure), and changes over time are difficult to quantify. The tracheal porcine pancreatic elastase model (PPE) produces severe injury, but the literature is conflicted as to whether emphysema improves, is stable, or progresses over time. We hypothesized a threshold of injury below which repair would occur and above which emphysema would be stable or progress. We treated 8-week-old C57BL6 mixed sex mice with 0, 0.5, 2, or 4 activity units of PPE in 100 µL PBS and performed lung stereology at 21 and 84 days. There were no significant differences in weight gain or mouse health. Despite minimal emphysema at 21-days in the 0.5 units group (2.8 µm increased mean linear intercept, MLI), MLI increased by 4.6 µm between days 21 and 84 (p = 0.0007). In addition to larger MLI at 21 days in 2- and 4-unit groups, MLI increases from day 21 to 84 were 17.2 and 34 µm respectively (p = 0.002 and p = 0.0001). Total lung volume increased, and alveolar surface area decreased with time and injury severity. Contrary to our hypothesis, we found no evidence of alveolar repair over time. Airspace destruction was both progressive and accelerative. Future mechanistic studies in lung immunity, mechano-biology, senescence, and cell-specific changes may lead to novel therapies to slow or halt progressive emphysema in humans.

Chymotrypsin-like Elastase-1 Mediates Progressive Emphysema in Alpha-1 Antitrypsin Deficiency.

Chymotrypsin-like elastase 1 (CELA1) is a serine protease that is neutralized by alpha-1antitrypsin (AAT) and prevents emphysema in a murine antisense oligonucleotide model of AAT-deficient emphysema. Mice with genetic ablation of AAT do not have emphysema at baseline but develop emphysema with injury and aging. We tested the role of the CELA1 gene in emphysema development in this genetic model of AAT-deficiency following tracheal lipopolysaccharide (LPS), 10 months of cigarette smoke exposure, aging, and a low-dose tracheal porcine pancreatic elastase (LD-PPE) model we developed. In this last model, we performed proteomic analysis to understand differences in lung protein composition. We were unable to show that AAT-deficient mice developed more emphysema than wild type with escalating doses of LPS. In the LD-PPE model, AAT-deficient mice developed significant and progressive emphysema from which Cela1-/- & AAT-deficient mice were protected. Cela1-/-& AAT-deficient lungs had more matrix-associated proteins than AAT-deficientlungs but also had more leukocyte-associated proteases. With cigarette smoke exposure, Cela1-/- &AAT-deficient mice had more emphysema than AAT-deficient mice but had less myeloperoxidase activity. Cela1-/-&AAT-deficient mice had less age-related airspace simplification than AAT-deficient and were comparable to wild type. While CELA1 promotes inflammation-independent emphysema progression and its absence preserves the lung matrix in multiple models of AAT-deficient emphysema, for unclear reasons Cela1 deficiency is associated with increased emphysema with cigarette smoke. While anti-CELA1 therapies could potentially be used to prevent emphysema progression in AAT deficiency after smoking cessation, an understanding of why and how cigarette smoke exacerbates emphysema in Cela1 deficiency and whether AAT replacement therapy mitigates this effect is needed first.

CELA1 Mediates Progressive Emphysema in Alpha-1 Antitrypsin Deficiency.

Chymotrypsin-like elastase 1 ( CELA1 ) is a serine protease that is neutralized by α1-antitrypsin (AAT) and prevents emphysema in a murine antisense oligonucleotide model of AAT-deficient emphysema. Mice with genetic ablation of AAT do not have emphysema at baseline but develop emphysema with injury and aging. We tested the role of CELA1 in emphysema development in this genetic model of AAT -deficiency following tracheal lipopolysacharide (LPS), 8 months of cigarette smoke (CS) exposure, aging, and a low-dose tracheal porcine pancreatic elastase (LD-PPE) model. In this last model, we performed proteomic analysis to understand differences in lung protein composition. We were unable to show that AAT -/ - mice developed more emphysema than wild type with LPS. In the LD-PPE model, AAT -/- mice developed progressive emphysema from which Cela1 -/- &AAT -/- mice were protected. In the CS model, Cela1 -/- &AAT -/- mice had worse emphysema than AAT -/- , and in the aging model, 72-75 week-old Cela1 -/- &AAT -/- mice had less emphysema than AAT -/- mice. Proteomic analysis of AAT -/- vs. wildtype lungs in the LD-PPE model showed reduced amounts of AAT proteins and increased amounts of proteins related to Rho and Rac1 GTPases and protein oxidation. Similar analysis of Cela1 -/- &AAT -/- vs. AAT -/- lungs showed differences in neutrophil degranulation, elastin fiber synthesis, and glutathione metabolism. Thus, Cela1 prevents post-injury emphysema progression in AAT -deficiency, but it has no effect and potentially worsens emphysema in response to chronic inflammation and injury. Prior to developing anti-CELA1 therapies for AAT-deficient emphysema, an understanding of why and how CS exacerbates emphysema in Cela1 deficiency is needed.

Multi-omic characterization of pediatric ARDS via nasal brushings.

RATIONALE: While nasal brushing transcriptomics can identify disease subtypes in chronic pulmonary diseases, it is unknown whether this is true in pediatric acute respiratory distress syndrome (PARDS). OBJECTIVES: Determine whether nasal transcriptomics and methylomics can identify clinically meaningful PARDS subgroups that reflect important pathobiological processes. METHODS: Nasal brushings and serum were collected on days 1, 3, 7, and 14 from control and PARDS subjects from two centers. PARDS duration was the primary endpoint. MEASUREMENTS AND MAIN RESULTS: Twenty-four control and 39 PARDS subjects were enrolled. Two nasal methylation patterns were identified. Compared to Methyl Subgroup 1, Subgroup 2 had hypomethylation of inflammatory genes and was enriched for immunocompromised subjects. Four transcriptomic patterns were identified with temporal patterns indicating injury, repair, and regeneration. Over time, both inflammatory (Subgroup B) and cell injury (Subgroup D) patterns transitioned to repair (Subgroup A) and eventually homeostasis (Subgroup C). When control specimens were included, they were largely Subgroup C. In comparison with 17 serum biomarkers, the nasal transcriptome was more predictive of prolonged PARDS. Subjects with initial Transcriptomic Subgroup B or D assignment had median PARDS duration of 8 days compared to 2 in A or C (p = 0.02). For predicting PARDS duration ≥ 3 days, nasal transcriptomics was more sensitive and serum biomarkers more specific. CONCLUSIONS: PARDS nasal transcriptome may reflect distal lung injury, repair, and regeneration. A combined nasal PCR and serum biomarker assay could be useful for predictive and diagnostic enrichment. Trial registration Clinicaltrials.gov NCT03539783 May 29, 2018.

Mouse lung organoid responses to reduced, increased, and cyclic stretch.

Most lung development occurs in the context of cyclic stretch. Alteration of the mechanical microenvironment is a common feature of many pulmonary diseases, with congenital diaphragmatic hernia (CDH) and fetal tracheal occlusion (FETO, a therapy for CDH) being extreme examples with changes in lung structure, cell differentiation, and function. To address limitations in cell culture and in vivo mechanotransductive models, we developed two mouse lung organoid (mLO) mechanotransductive models using postnatal day 5 (PND5) mouse lung CD326-positive cells and fibroblasts subjected to increased, decreased, and cyclic strain. In the first model, mLOs were exposed to forskolin (FSK) and/or disrupted (DIS) and evaluated at 20 h. mLO cross-sectional area changed by +59%, +24%, and -68% in FSK, control, and DIS mLOs, respectively. FSK-treated organoids had twice as many proliferating cells as other organoids. In the second model, 20 h of 10.25% biaxial cyclic strain increased the mRNAs of lung mesenchymal cell lineages compared with static stretch and no stretch. Cyclic stretch increased TGF-ß and integrin-mediated signaling, with upstream analysis indicating roles for histone deacetylases, microRNAs, and long noncoding RNAs. Cyclic stretch mLOs increased αSMA-positive and αSMA-PDGFRα-double-positive cells compared with no stretch and static stretch mLOs. In this PND5 mLO mechanotransductive model, cell proliferation is increased by static stretch, and cyclic stretch induces mesenchymal gene expression changes important in postnatal lung development.

Transuterine Fetal Tracheal Occlusion Model in Mice.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Fetal Tracheal Occlusion Increases Lung Basal Cells via Increased Yap Signaling.

Fetal endoscopic tracheal occlusion (FETO) is an emerging surgical therapy for congenital diaphragmatic hernia (CDH). Ovine and rabbit data suggested altered lung epithelial cell populations after tracheal occlusion (TO) with transcriptomic signatures implicating basal cells. To test this hypothesis, we deconvolved mRNA sequencing (mRNA-seq) data and used quantitative image analysis in fetal rabbit lung TO, which had increased basal cells and reduced ciliated cells after TO. In a fetal mouse TO model, flow cytometry showed increased basal cells, and immunohistochemistry demonstrated basal cell extension to subpleural airways. Nuclear Yap, a known regulator of basal cell fate, was increased in TO lung, and Yap ablation on the lung epithelium abrogated TO-mediated basal cell expansion. mRNA-seq of TO lung showed increased activity of downstream Yap genes. Human lung specimens with congenital and fetal tracheal occlusion had clusters of subpleural basal cells that were not present in the control. TO increases lung epithelial cell nuclear Yap, leading to basal cell expansion.

Sex-specific differences in emphysema using a murine antisense oligonucleotide model of α-1 antitrypsin deficiency.

α-1 Antitrypsin (AAT) deficiency is the leading genetic cause of emphysema; however, until recently, no genuine animal models of AAT deficiency existed, hampering the development of new therapies. This shortcoming is now addressed by both AAT-null and antisense oligonucleotide mouse models. The goal of this study was to more fully characterize the antisense oligonucleotide model. Both liver AAT mRNA and serum AAT levels were lower in anti-AAT versus control oligonucleotide-treated mice after 6, 12, and 24 wk. Six and twelve weeks of anti-AAT oligonucleotide therapy induced emphysema that was worse in female than male mice: mean linear intercept 73.4 versus 62.5 µm (P = 0.000003). However, at 24 wk of treatment, control oligonucleotide-treated mice also developed emphysema. After 6 wk of therapy, anti-AAT male and female mice demonstrated a similar reduction serum AAT levels, and there were no sex or treatment-specific alterations in inflammatory, serine protease, or matrix metalloproteinase mRNAs, with the exception of chymotrypsin-like elastase 1 (Cela1), which was 7- and 9-fold higher in anti-AAT versus control male and female lungs, respectively, and 1.6-fold higher in female versus male anti-AAT-treated lungs (P = 0.04). While lung AAT protein levels were reduced in anti-AAT-treated mice, lung AAT mRNA levels were unaffected. These findings are consistent with increased emphysema susceptibility of female patients with AAT-deficiency. The anti-AAT oligonucleotide model of AAT deficiency is useful for compartment-specific, in vivo molecular biology, and sex-specific studies of AAT-deficient emphysema, but it should be used with caution in studies longer than 12-wk duration.

Proteomic profiling of tracheal fluid in an ovine model of congenital diaphragmatic hernia and fetal tracheal occlusion.

Congenital diaphragmatic hernia (CDH) occurs in ~1:2,000 pregnancies and is associated with substantial morbidity and mortality. Fetal tracheal occlusion (TO) is an emerging therapy that improves lung growth and reduces mortality, although substantial respiratory compromise persists in survivors. In this study, we used tracheal fluid in a fetal sheep model of CDH with TO for proteomic analysis with subsequent validation of findings in sheep lung tissue. We found that the proteomic profiles of CDH tracheal fluid was most similar to control lung and CDH/TO lung most similar to TO lung. Among 118 proteins altered in CDH, only 11 were reciprocally regulated in CDH/TO. The most significantly altered pathways and processes were cell proliferation, phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling, inflammation, and microtubule dynamics. CDH suppressed and TO promoted cell proliferation and AKT-related signaling cascades. By Western blot analysis and immunohistochemistry, epithelial PCNA and phosphorylated AKT were decreased in CDH and increased in TO and CDH/TO lungs. The Wnt target Axin2 was decreased threefold in CDH lung compared with control without a significant increase in CDH/TO lung. Cilia-related pathways were among the most dysregulated with CDH lung having a nearly twofold increase in acetylated α-tubulin and a relative increase in the number of ciliated cells. While TO improves lung growth and patient survival in CDH, the procedure substantially alters many processes important in lung development and cell differentiation. Further elucidation of these changes will be critical to improving lung health in infants with CDH treated with TO.

Fetal tracheal occlusion in mice: a novel transuterine method.

BACKGROUND: Fetal tracheal occlusion (TO) is an emerging surgical therapy in congenital diaphragmatic hernia that improves the fetal lung growth. Different animal models of congenital diaphragmatic hernia and TO present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and available genetic tools. We developed a minimally invasive murine transuterine TO model, which will be useful in defining how TO impacts lung molecular biology, cellular processes, and overall lung physiology. MATERIALS AND METHODS: Time-mated C57BL/6 mice underwent laparotomy at embryonic day 16.5 (E16.5) with transuterine TO performed on two fetuses in each uterine horn. At E18.5, dams were sacrificed and fetuses harvested. The lungs of the TO fetuses were compared with the nonmanipulated counterparts by morphometric and histologic analysis. RESULTS: Successful TO was confirmed in 16 of 20 TO fetuses. Twelve of them survived to E18.5 (75%). Fetal weights were comparable, but lung weights were significantly greater in TO (28.41 ± 5.87 versus 23.38 ± 3.09, P = 0.043). Lung to body weight ratio was also greater (0.26 ± 0.003 versus 0.22 ± 0.002, P = 0.006). E18.5 TO lungs demonstrated dilated central and distal airspaces with increased cellularity. DNA/protein and DNA/lung weight ratios were elevated while protein/lung weight ratio was lower in TO compared to control. CONCLUSIONS: Mice fetal transuterine TO is feasible with comparable outcomes to other current animal models. The increase in the lung weight, lung to body weight ratio and the DNA/protein ratio indicate organized lung growth rather than edema or cell hypertrophy.

Role for Cela1 in Postnatal Lung Remodeling and Alpha-1 Antitrypsin-Deficient Emphysema.

Alpha-1 antitrypsin (AAT) deficiency-related emphysema is the fourth leading indication for lung transplant. Chymotrypsin-like elastase 1 (Cela1) is a digestive protease that is expressed during lung development in association with regions of elastin remodeling, exhibits stretch-dependent expression during lung regeneration, and binds lung elastin in a stretch-dependent manner. AAT covalently neutralizes Cela1 in vitro. We sought to determine the role of Cela1 in postnatal lung physiology, whether it interacted with AAT in vivo, and to detect any effects it may have in the context of AAT deficiency. The lungs of Cela1-/- mice had aberrant lung elastin structure and higher elastance as assessed with the flexiVent system. On the basis of in situ zymography with ex vivo lung stretch, Cela1 was solely responsible for stretch-inducible lung elastase activity. By mass spectrometry, Cela1 degraded mature elastin similarly to pancreatic elastase. Cela1 promoter and protein sequences were phylogenetically distinct in the placental mammal lineage, suggesting an adaptive role for lung-expressed Cela1 in this clade. A 6-week antisense oligonucleotide mouse model of AAT deficiency resulted in emphysema with increased Cela1 mRNA and reduction of approximately 70 kD Cela1, consistent with covalent binding of Cela1 by AAT. Cela1-/- mice were completely protected against emphysema in this model. Cela1 was increased in human AAT-deficient emphysema. Cela1 is important in physiologic and pathologic stretch-dependent remodeling processes in the postnatal lung. AAT is an important regulator of this process. Our findings provide proof of concept for the development of anti-Cela1 therapies to prevent and/or treat AAT-deficient emphysema.

Excessive Reversal of Epidermal Growth Factor Receptor and Ephrin Signaling Following Tracheal Occlusion in Rabbit Model of Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) causes severe pulmonary hypoplasia from herniation of abdominal contents into the thorax. Tracheal occlusion (TO) for human CDH improves survival, but morbidity and mortality remain high, and we do not fully understand the cellular pathways and processes most severely impacted by CDH and TO. We created a left diaphragmatic hernia (DH) in rabbit fetuses with subsequent TO and collected left lung sections for NextGen mRNA sequencing. DH, TO, and DHTO fetuses had comparable body and organ growth to control except for lower lung weights in DH (p<0.05). Of 13,687 expressed genes, DHTO had 687 differentially expressed genes compared to DH, but no other group-group comparison had more than 10. Considering genes in combination, many of the genes reduced in DH were more highly expressed in DHTO than in control. Benchmarking fetal rabbit lung gene expression to published lung development data, both DH and DHTO lungs were more highly correlated with the gene expression of immature lung. DNA synthesis was upregulated in DHTO compared to DH and ribosome and protein synthesis pathways were downregulated. DH reduced total and epithelial cell proliferation by half and two-thirds respectively, and DHTO increased proliferation by 2.5 and 3.4-fold respectively. Signaling pathways downregulated by DH and upregulated in DHTO were epidermal growth factor receptor signaling, ephrin signaling, and cell migration; however, levels of ephrin and EGFR signaling in DHTO exceeded that of control. Identification and inhibition of the ligands responsible for this dysregulated signaling could improve lung development in CDH.

Stretch regulates expression and binding of chymotrypsin-like elastase 1 in the postnatal lung.

Lung stretch is critical for normal lung development and for compensatory lung growth after pneumonectomy (PNX), but the mechanisms by which strain induces matrix remodeling are unclear. Our prior work demonstrated an association of chymotrypsin-like elastase 1 (Cela1) with lung elastin remodeling, and that strain triggered a near-instantaneous elastin-remodeling response. We sought to determine whether stretch regulates Cela1 expression and Cela1 binding to lung elastin. In C57BL/6J mice, Cela1 protein increased 176-fold during lung morphogenesis. Cela1 was covalently bound to serpin peptidase inhibitor, clade A, member 1, resulting in a higher molecular mass in lung homogenate compared to pancreas homogenate. Post-PNX, Cela1 mRNA increased 6-fold, protein 3-fold, and Cela1-positive cells 2-fold. Cela1 was expressed predominantly in alveolar type II cells in the embryonic lung and predominantly in CD90-positive lung fibroblasts postnatally. During compensatory lung growth, Cela1 expression was induced in nonproliferative mesenchymal cells. In ex vivo mouse lung sections, stretch increased Cela1 binding to lung tissue by 46%. Competitive inhibition with soluble elastin completely abrogated this increase. Areas of stretch-induced elastase activity and Cela1 binding colocalized. The stretch-dependent expression and binding kinetics of Cela1 indicate an important role in stretch-dependent remodeling of the peripheral lung during development and regeneration.

HER2-mTOR signaling-driven breast cancer cells require ER-associated degradation to survive.

Targeting non-oncogenic vulnerabilities may provide additional therapeutic approaches in tumors that are resistant to oncogene-targeted therapy. Using a computational pathway-based approach, we interrogated clinical breast cancer genomic data sets for candidate non-oncogenic vulnerabilities in breast cancers that have genomic amplification of ERBB2, which encodes human epidermal growth factor 2 (HER2). HER2-positive (HER2(+)) breast cancers showed increased expression of genes encoding proteins in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Genetic ablation or pharmacological inhibition of ERAD led to irrecoverable ER stress and selectively killed HER2(+) breast cancer cells. Cell death caused by ERAD inhibition partially depended on increased HER2-mTOR signaling, which imposed an increased proteotoxic burden on the ER. Cell death in response to ER stress required the IRE1α-JNK pathway, which was selectively suppressed in HER2(+) breast cancers by phosphatases that inactivate JNK. Accordingly, the cytotoxicity of inhibiting ERAD as well as JNK phosphatases was synergistic in HER2(+) but not in HER2-negative breast cancer cells. Therefore, our study suggests that reactivation of oncogene-induced stress by targeting stress-adaptive pathways may be a beneficial approach for therapy-resistant breast cancers.

Localization and stretch-dependence of lung elastase activity in development and compensatory growth.

Synthesis and remodeling of the lung matrix is necessary for primary and compensatory lung growth. Because cyclic negative force is applied to developing lung tissue during the respiratory cycle, we hypothesized that stretch is a critical regulator of lung matrix remodeling. By using quantitative image analysis of whole-lung and whole-lobe elastin in situ zymography images, we demonstrated that elastase activity increased twofold during the alveolar stage of postnatal lung morphogenesis in the mouse. Remodeling was restricted to alveolar walls and ducts and was nearly absent in dense elastin band structures. In the mouse pneumonectomy model of compensatory lung growth, elastase activity increased threefold, peaking at 14 days postpneumonectomy and was higher in the accessory lobe compared with other lobes. Remodeling during normal development and during compensatory lung growth was different with increased major airway and pulmonary arterial remodeling during development but not regeneration, and with homogenous remodeling throughout the parenchyma during development, but increased remodeling only in subpleural regions during compensatory lung growth. Left lung wax plombage prevented increased lung elastin during compensatory lung growth. To test whether the adult lung retains an innate capacity to remodel elastin, we developed a confocal microscope-compatible stretching device. In ex vivo adult mouse lung sections, lung elastase activity increased exponentially with strain and in peripheral regions of lung more than in central regions. Our study demonstrates that lung elastase activity is stretch-dependent and supports a model in which externally applied forces influence the composition, structure, and function of the matrix during periods of alveolar septation.

Phosphodiesterase (PDE) inhibitor torbafylline (HWA 448) attenuates burn-induced rat skeletal muscle proteolysis through the PDE4/cAMP/EPAC/PI3K/Akt pathway.

Treatment of rats after burn-injury with the cyclic AMP phosphodiesterase (PDE) inhibitor, torbafylline (also known as HWA 448) significantly reversed changes in rat skeletal muscle proteolysis, PDE4 activity, cAMP concentrations and mRNA expression of TNFα, IL-6, ubiquitin and E3 ligases. Torbafylline also attenuated muscle proteolysis during in vitro incubation, and this effect was blocked by the inhibitor Rp-cAMPS. Moreover, torbafylline significantly increased phospho-Akt levels, and normalized downregulated phospho-FOXO1 and phospho-4E-BP1 in muscle of burn rats. Similarly, torbafylline also normalized phosphorylation levels of Akt and its downstream elements in TNFα+IFNγ treated C2C12 myotubes. Torbafylline enhanced protein levels of exchange protein directly activated by cAMP (Epac) both in skeletal muscle of burn rats and in TNFα+IFNγ treated C2C12 myotubes. Pretreatment with a specific antagonist of PI3K or Epac significantly reversed the inhibitory effects of torbafylline on TNFα+IFNγ-induced MAFbx mRNA expression and protein breakdown in C2C12 myotubes. Torbafylline inhibits burn-induced muscle proteolysis by activating multiple pathways through PDE4/cAMP/Epac/PI3K/Akt.

Des-acyl ghrelin exhibits pro-anabolic and anti-catabolic effects on C2C12 myotubes exposed to cytokines and reduces burn-induced muscle proteolysis in rats.

Although ghrelin and GHRP-2 have been shown to inhibit skeletal muscle proteolysis in rats with burn injury, the effects of des-acyl ghrelin (DAG) have not been reported. In this paper, we demonstrate that continuous 24h administration of DAG attenuated burn-induced EDL muscle proteolysis, and normalized elevated TNFα mRNA. Combined treatment of cultured C2C12 myotubes with TNFα and IFN-γ (TNF+IFN) inhibited protein synthesis and increased protein breakdown; DAG abolished both effects. PI3 kinase inhibition by LY294002 and mTOR inhibition by rapamycin blocked the reversal of the anti-anabolic effects of TNF+IFN-treated myotubes by DAG. DAG also reversed or attenuated the TNF+IFN-induced reduction in phosphorylation of Akt, FOXO1, 4E-BP-1, and GSK-3ß in myotubes. Furthermore, DAG attenuated the atrophy signal, phospho-NF-κB, and the mRNA expression of MAFbx and MuRF1, upregulated by TNF+IFN in C2C12 myotubes. We conclude that DAG reduces muscle cachexia produced by injury and proinflammatory cytokines, and that DAG or DAG-based compounds may be useful in treating wasting disorders.

Ghrelin receptor agonist, GHRP-2, attenuates burn injury-induced MuRF-1 and MAFbx expression and muscle proteolysis in rats.

Thermal injury results in hypermetabolism, loss of body weight, and skeletal muscle wasting in mice and rats. Our earlier studies have demonstrated that ghrelin injection stimulates food intake and growth hormone release and inhibits skeletal muscle proteolysis in rats with thermal injury. We sought to develop a lower molecular weight, stable and longer acting peptide to combat the catabolic responses caused by thermal injury. Towards this goal, we examined the role of the hexapeptide mimetic of ghrelin, growth hormone-releasing peptide-2 (GHRP-2), on expression of E3 ubiquitin ligases and breakdown of muscle protein in rats with thermal injury. Slow in vivo release of GHRP-2 through minipump for 24h attenuated the thermal injury-induced increase in mRNA expression of IL-6 and of the E3 ubiquitin ligases, MuRF-1 and MAFbx, in rat skeletal muscle. Furthermore, burn-induced increases in total and myofibrillar protein breakdown from rat EDL muscle were attenuated by GHRP-2. These findings suggest that catabolic responses resulting from thermal injury can be attenuated by GHRP-2.