Agouti Mice Models to Study the Diabetogenicity of FK506 (Tacrolimus)

Dominant mutations in the promoter region of the mouse agouti gene lead to a syndrome characterized by agouti yellow coat color, obesity, hyperinsulinemia, hyperglycemia, type II diabetes and increased linear growth. The mechanism of agouti modulation of the coat color development as well as obesity involves competitive antagonism of the ? -melanocortin stimulating hormone at its receptor. Hyperplasia of the pancreatic ? cells in agouti mice precedes the development of obesity. Enhanced insulin secretion is induced in vitro by agouti effects on calcium influx into islet cells. Preliminary testing of insulin producing pancreatic cells has shown that exposure to high doses of an immunosuppressive drug tacrolimus (FK506) altered the secretion of insulin. The hypothesis of the present study is that FK506 treatment of transgenic mice, which either overexpress the agouti gene ubiquitously or in the adipose tissue, may alter expression of agouti mRNA and protein. Agouti mice also have an impairment of the immune system thus, another hypothesis to be tested was if immunosuppression by FK506 would amplify the agouti phenotype. There was decrease in food intake and glucose levels in the aP2 mice with Tacrolimus (FK506). There was no detection of agouti mRNA or protein in adipose or pancreatic tissue of wild type mice and no significant effect of FK506 on agouti mRNA or protein expression in the aP2 mice. Toxic effects of FK506 at this dose in both mice were not significant

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

Basal cell; Fetal tracheal occlusion; MechanotransductionCélula basal; Oclusión traqueal fetal; MecanotransducciónCèl·lula basal; Oclusió traqueal fetal; MecanotransduccióFetal 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.Funding was obtained from NIH/NHLBI R01HL141229 (to BV)

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

Abstract 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

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

Abstract 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 with 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 with 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

Role for Cela1 in Postnatal Lung Remodeling and AAT-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