Sherry Mabry

Chronic hypoxia and rat lung development: analysis by morphometry and directed microarray.

It is unclear how sublethal hypoxia affects lung development. To investigate the effects of chronic hypoxia on postnatal lung remodeling, we treated neonatal rats with FIO2 of 0.12 for 10 d and analyzed lung development by morphometry and gene expression by DNA microarray. Our results showed the neonatal rats exposed to hypoxia reduced body weight by 42% and wet lung weight by 32% compared with the neonatal rats exposed to normoxia. In the neonatal rats exposed to hypoxia, the radial alveolar counts were decreased to 5.6 from 7.9 and the mean linear intercepts were increased to 56.5 μm from 38.2 μm. In DNA microarray analysis, approximately half of probed genes were unknown. Chronic hypoxia significantly regulated expression of genes that are involved in pathogenesis of pulmonary hypertension and postnatal lung remodeling. Chemokine ligand 12, jagged 2 were among those upregulated; c-kit, ephrin A1, and Hif-2α were among those downregulated. The altered expression of those genes was correlated with the lung development and remodeling.

High-dose inhaled nitric oxide and hyperoxia increases lung collagen accumulation in piglets

Nitric oxide (NO), a pro-oxidant gas, is used with hyperoxia (O2) to treat neonatal pulmonary hypertension and recently bronchopulmonary dysplasia, but great concerns remain regarding NO’s potential toxicity. Based on reports that exposure to oxidant gases results in pulmonary extracellular matrix injury associated with elevated lavage fluid levels of extracellular matrix components, we hypothesized that inhaled NO with or without hyperoxia will have the same effect. We measured alveolar septal width, lung collagen content, lavage fluid hydroxyproline, hyaluronan and laminin levels in neonatal piglets after 5 days’ exposure to room air (RA), RA + 50 ppm NO (RA + NO), O2 (FiO2 > 0.96) or O2 + NO. Matrix metalloproteinase (MMP) activity and MMP-2 mRNA were also measured. In recovery experiments, we measured lung collagen content in piglets exposed to RA + NO or O2 + NO and then allowed to recover for 3 days. The results show that lung collagen increased 4-fold in the RA + NO piglets, the O2 and O2 + NO groups had only a 2-fold elevation relative to RA controls. Unlike the RA + NO piglets, the O2 and O2 + NO groups had more than 20-fold elevation in lung lavage fluid hydroxyproline compared to the RA group. O2 and O2 + NO also had increased lung MMP activity, extravascular water, and lavage fluid proteins. MMP-2 mRNA levels were unchanged. After 3 days’ recovery in room air, the RA + NO groups’ lung collagen had declined from 4-fold to 2-fold above the RA group values. The O2 + NO group did not decline. Alveolar septal width increased significantly only in the O2 and O2 + NO groups. We conclude that 5 days’ exposure to NO does not result in pulmonary matrix degradation but instead significantly increases lung collagen content. This effect appears potentially reversible. In contrast, hyperoxia exposure with or without NO results in pulmonary matrix degradation and increased lung collagen content. The observation that NO increased lung collagen content represents a new finding and suggests NO could potentially induce pulmonary fibrosis.

Airway muscle in infants with congenital diaphragmatic hernia: Response to treatment

Abstract Background/Purpose: Airway muscle hyperactivity and chronic lung disease frequently follow congenital diaphragmatic hernia (CDH) treatment. The aim of this study was to compare the quantity of airway muscle and alveolar ductal artery muscle in CDH infants after various treatments. Methods: Five groups were studied postmortem: CDH, died within 24 hours, without high ventilatory assistance ( n = 3); CDH, various extracorporeal membrane oxygenation (ECMO) durations, without high ventilatory assistance ( n = 4); CDH, various ECMO durations, with high ventilatory assistance ( n = 7); no CDH, without high ventilatory assistance ( n = 12); and no CDH, with high ventilatory assistance and bronchopulmonary dysplasia (BPD) ( n = 5). Sections from standardized fixed lungs were immunohistochemically stained for α-smooth muscle actin. Muscle surrounding conducting airways from small preterminal bronchioles to bronchi was quantitated in both the ipsilateral and contralateral lungs with computerized image analysis. Similarly, muscle mass was quantitated in alveolar ductal arteries. Results: CDH infants with low ventilatory assistance, regardless of postnatal age, had the same quantity of airway muscle as low ventilatory assistance controls. Infants with CDH and prolonged high ventilatory assistance had significantly more muscle throughout the conducting airways, similar to BPD infants without CDH, even though the CDH infants had significantly less exposure to high ventilatory assistance. With both low and high ventilatory assistance, the quantity of muscle in both the ipsilateral and contralateral lungs was similar. In contrast, small acinar arteries in CDH infants have increased muscle mass at birth. This muscle is decreased by ECMO but persists in CDH infants with high ventilatory assistance. Conclusions: The authors show that postnatally, CDH infants acquire increased muscle quantity throughout the conducting airways, in both the ipsilateral and contralateral lungs, with relatively short exposure to high ventilatory assistance. The normal decrease in acinar arterial mass that occurs postnatally is delayed in CDH infants with high ventilatory assistance.

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation

BackgroundMyofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFβ receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts—a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known.MethodsTRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis.ResultsThe down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFβ ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis.ConclusionsTRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.

Lung Antioxidant Enzymes and Cardiopulmonary Responses in Young Rats Exposed to Hyperoxia and Treated Intratracheally with PEG Catalase and Superoxide Dismutase

The 27-day-old rat exposed to 100% oxygen (O2) for 8 days will have predictable lung vascular and parenchymal changes at 60 days of age. Using this model, the goals of this study are (1) to measure the lung antioxidant enzyme activities serially following intratracheal PEG antioxidant therapy during the 8-day O2 exposure; and (2) to assess chronic cardiopulmonary changes in the O2-exposed rats treated with PEG-CAT and/or PEG-CuZn SOD given intraperitoneally (IP) and/or intratracheally (IT). The study encompassed 202 male rats exposed to room air or oxygen. CuZn SOD doses were 300 U IT and 2000 U IP. The CAT dose was 500 or 4000 U IT and 10,000 U IP. At 60 days of age, the right ventricular systolic pressure (RVP), RV weight, % acinar wall arterial thickness, and parenchymal air space were significantly increased in O2-exposed rats compared to RA rats. The RVP, RV weight, and parenchymal changes were prevented by daily IT PEG-CAT 4000 U + CuZn SOD 300 U but the increased small artery muscularization was not. Three hours after the initial dose of IT PEG-CAT 4000 U, lung CAT activity was more than doubled and remained constant throughout the 8-day daily treatment course. This dose of CAT depressed the induction response to O2 of CuZn and MnSOD. It is concluded that daily intratracheal administration of PEG-CAT 4000 U + CuZn SOD 300 U can significantly ameliorate some of the chronic parenchymal and vascular lung O2 toxic changes. However, it appears that high-dose exogenous PEG-CAT suppresses the endogenous enzyme induction to hyperoxia of both CuZn and Mn-SOD.

Lung epithelial‐specific TRIP‐1 overexpression maintains epithelial integrity during hyperoxia exposure

The onset and degree of injury occurring in animals that develop hyperoxic acute lung injury (HALI) is dependent on age at exposure, suggesting that developmentally regulated pathways/factors must underlie initiation of the epithelial injury and subsequent repair. Type II TGFβ receptor interacting protein‐1 (TRIP‐1) is a negative regulator of TGFβ signaling, which we have previously shown is a developmentally regulated protein with modulatory effects on epithelial‐fibroblastic signaling. The aim of this study was to assess if type II alveolar epithelial cells overexpressing TRIP‐1 are protected against hyperoxia‐induced epithelial injury, and in turn HALI. Rat lung epithelial cells (RLE) overexpressing TRIP‐1 or LacZ were exposed to 85% oxygen for 4 days. A surfactant protein C (SPC)‐driven TRIP‐1 overexpression mouse (TRIP‐1AECTg+) was generated and exposed to hyperoxia (>95% for 4 days) at 4 weeks of age to assess the effects TRIP‐1 overexpression has on HALI. RLE overexpressing TRIP‐1 resisted hyperoxia‐induced apoptosis. Mice overexpressing TRIP‐1 in their lung type II alveolar epithelial cells (TRIP‐1AECTg+) showed normal lung development, increased phospho‐AKT level and E‐cadherin, along with resistance to HALI, as evidence by less TGFβ activation, apoptosis, alveolar macrophage influx, KC expression. Taken together, these findings point to existence of a TRIP‐1 mediated molecular pathway affording protection against epithelial/acute lung injury.

Expression of Epidermal Growth Factor-Like Domain 7 in Neonatal Rat Lungs During Normoxia and Hyperoxia.

Purpose of Study: Preterm babies treated with ventilator support and supplemental oxygen frequently develop chronic lung disease (CLD) that has significant mortality and morbidity. Oxygen toxicity plays an important role in CLD etiology. Several lines of evidence have suggested that impairment of pulmonary angiogenesis is implicated in alveolization and the development of CLD. Epidermal growth factor-like domain 7 (EGFL-7) is a recently identified protein secreted from vascular endothelial cells and it regulates vascular tubulogenesis (Nature 2004;428:754). Aim of this study was to measure EGFL-7 expression in the neonatal lung during normoxia and hyperoxia.Methods: Rat pups at 4 days of age were randomly assigned to normoxic and hyperoxic groups. The rats in the normoxic and hyperoxic groups were treated with room air and 95% O2 for 3, 6, and 10 days, respectively. The lung tissues were collected for total RNA isolation. EGFL-7 mRNA expression was measured by quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR). Separately, human umbilical vein endothelial cells (HUVEC) were cultured in 37°C, 5% CO2 incubator, and were exposed to normoxia (room air) or hyperoxia (95% O2).Results EGFL-7 mRNA in normoxic neonatal rat lung was consistently expressed from 7 days to 2 months of age (n = 3) at each time. EGFL-7 mRNA expression in the hyperoxic group was significantly decreased after oxygen exposure for 3, 6 and 10 days; it decreased 2.1 fold at day 3 (n = 3); 4.1 fold at day 6 (n = 3); and 3.1 fold at day 10 (n = 3) compared to time-matched normoxic group results, respectively. EGFL-7 mRNA expression in the hyperoxic group returned to nearly normal levels 2 weeks (n = 3) after discontinuing oxygen exposure, and it remained at normal levels during the 2 month recovery period (n = 2–3). In cultured HUVEC, EGFL-7 mRNA expression also decreased 2.6 fold after 95% O2 exposure for 48 hours.Conclusions: Oxygen exposure is associated with the decrease of EGFL-7 mRNA expression in the neonatal rat lung and the expression level returns to normal after oxygen treatment. These findings imply that reduced levels of EGFL-7 at a critical lung development stage may be a contributing factor in the impairment of pulmonary angiogenesis and alveolization after hyperoxic lung injury.