Increase In Alveolar Number Research Articles

Bufei Yishen Formula Restores Th17/Treg Balance and Attenuates Chronic Obstructive Pulmonary Disease via Activation of the Adenosine 2a Receptor

Bufei Yishen formula (BYF) is a Traditional Chinese Medicine (TCM) reported to ameliorate chronic obstructive pulmonary disease (COPD) by regulating the balance between T helper (Th) 17 and regulatory T (Treg) cells. However, its mechanism remains unknown. Therefore, this study aimed to explore the underlying mechanisms of BYF. Naïve CD4+ T cells were exposed to anti-CD3, anti-CD28, transforming growth factor (TGF)-β, and/or interleukin (IL)-6 to promote their differentiation into Th17 or Treg cells. A rat model of cigarette smoke- and bacterial infection-induced COPD was established and orally treated with BYF and/or an adenosine 2a receptor (A2aR) antagonist. Then, the rats were sacrificed, their lung tissues were removed for histological analysis, and their spleens were collected to evaluate Th17 and Treg cells. The results showed that BYF significantly suppressed Th17 cell differentiation and its related cytokines and enhanced Treg cell differentiation and its related cytokines. In addition, BYF activated the A2aR, increased the levels of p-signal transducer and activator of transcription (STAT)5, and decreased the level of p-STAT3 in Treg and Th17 cells. The A2aR antagonist suppressed the changes induced by BYF treatment in Th17 and Treg cells. Furthermore, the A2aR antagonist diminished the therapeutic effect of BYF on COPD, as indicated by the lung injury scores, bronchiole wall thickness, small pulmonary vessels wall thickness, bronchiole stenosis, alveolar diameters, decrease in inflammatory cytokines, increase in alveolar number, and lung functions. Similarly, the A2aR antagonist reversed the effects of BYF on the proportion of Th17 and Treg cells in the spleen. Additionally, BYF increased the protein and mRNA levels of A2aR and regulated the phosphorylation of STAT3 and STAT5 in spleen and lung tissues, which were inhibited by cotreatment with the A2aR antagonist. In conclusion, this study suggested that BYF exhibited its anti-COPD efficacy by restoring the Th17/Treg balance via activating A2aR, which may provide evidence for the clinical application of BYF in the treatment of COPD.

Rat lungs show a biphasic formation of new alveoli during postnatal development.

Roughly 90% of the gas-exchange surface is formed by alveolarization of the lungs. To the best of our knowledge, the formation of new alveoli has been followed in rats only by means of morphological description or interpretation of semiquantitative data until now. Therefore, we estimated the number of alveoli in rat lungs between postnatal days 4 and 60 by unambiguously counting the alveolar openings. We observed a bulk formation of new alveoli between days 4 and 21 (17.4 times increase from 0.8 to 14.3 millions) and a second phase of continued alveolarization between days 21 and 60 (1.3 times increase to 19.3 million). The (number weighted) mean volume of the alveoli decreases during the phase of bulk alveolarization from ∼593,000 μm(3) at day 4 to ∼141,000 μm(3) at day 21, but increases again to ∼298,000 μm(3) at day 60. We conclude that the "bulk alveolarization" correlates with the mechanism of classical alveolarization (alveolarization before the microvascular maturation is completed) and that the "continued alveolarization" follows three proposed mechanisms of late alveolarization (alveolarization after microvascular maturation). The biphasic pattern is more evident for the increase in alveolar number than for the formation of new alveolar septa (estimated as the length of the free septal edge). Furthermore, a striking negative correlation between the estimated alveolar size and published data on retention of nanoparticles was detected.

Good News for Lung Repair in Preterm Infants

In this issue of the Journal, Narayanan and colleagues (pp. 1104–1109) demonstrated with hyperpolarized helium nuclear magnetic resonance (NMR) (3HeNMR) that the alveolar number and size in preterm infants who had bronchopulmonary dysplasia (BPD) were normal at 10 to 14 years (1). This good news will change how we think about the growth and repair of the injured preterm lung. There is a developmental context for appreciating this new result. The fetal human airways branch about 17 times by about 18 weeks of gestation. A further six generations of airway branching forms the bronchioles and alveolar ducts to yield 500,000 saccules by about 32 to 34 weeks gestation (2). Amazingly, the saccular preterm human lung at 24 weeks gestation can provide gas exchange, and saccular divisions can continue until alveolarization begins. Very preterm lungs also can have altered development from antenatal exposures such as maternal smoking, fetal growth restriction, and chorioamnionitis. The preterm lung is easily injured by mechanical ventilation, oxygen, infection, and probably nutritional deficits despite the best efforts of caretakers. Although survival of these infants has increased strikingly in recent years from advances such as antenatal corticosteroids, postnatal surfactant, and more gentle approaches to mechanical ventilation, the incidence of BPD remains over 40% for infants born at 22 to 28 weeks gestation who survive to 36 weeks gestation (3).

Ozone Exposure During thE Early Postnatal Period Alters the Timing and Pattern of Alveolar Growth and Development in Nonhuman Primates

Exposure to oxidant air pollutants in early childhood, with ozone as the key oxidant, has been linked to significant decrements in pulmonary function in young adults and exacerbation of airway remodeling in asthma. Development of lung parenchyma in rhesus monkeys is rapid during the first 2 years of life (comparable to the first 6 years in humans). Our hypothesis is that ozone inhalation during infancy alters alveolar morphogenesis. We exposed infant rhesus monkeys biweekly to 5, 8 hr/day, cycles of 0.5 ppm ozone with or without house dust mite allergen from 1 to 3 or 1 to 6 months of age. Monkeys were necropsied at 3 and 6 months of age. A morphometric approach was used to quantify changes in alveolar volume and number, the distribution of alveolar size, and capillary surface density per alveolar septa. Quantitative real time PCR was used to measure the relative difference in gene expression over time. Monkeys exposed to ozone alone or ozone combined with allergen had statistically larger alveoli that were less in number at 3 months of age. Alveolar capillary surface density was also decreased in the ozone exposed groups at 3 months of age. At 6 months of age, the alveolar number was similar between treatment groups and was associated with a significant rise in alveolar number from 3 to 6 months of age in the ozone exposed groups. This increase in alveolar number was not associated with any significant increase in microvascular growth as measured by morphometry or changes in angiogenic gene expression. Inhalation of ozone during infancy alters the appearance and timing of alveolar growth and maturation. Understanding the mechanism involved with this altered alveolar growth may provide insight into the parenchymal injury and repair process that is involved with chronic lung diseases such as severe asthma and COPD.

NOS2 mediates lung structure and function changes in the SP-D model of emphysema without improving surfactant homeostasis

The collectin Surfactant protein D (SP-D) is an innate host defense protein with well-established immuno-modulatory properties. Mice deficient in SP-D (SP-D−/−) spontaneously develop typical emphysematous and inflammatory alterations: a significant decrease in alveolar number, accumulation of enlarged alveolar macrophages overproduction of the inducible nitric oxide synthase (iNOS), hypertrophy and hyperplasia of alveolar type II cells and disturbances of surfactant homoeostasis. Using design-based stereology, wild type, iNOS(−/−), SP-D(−/−) and double knockout (DiNOS) mice at 12 weeks of age were measured for a series of structural parameters. Lung resistance and elastance as functions of frequency at positive end-expiratory pressures in the range of 0–9 cm H2O were measured using Flexivent. The additional ablation of the iNOS gene improved emphysematous alterations in the SP-D(−/−) mouse resulting in a significant increase in alveolar number and a reduction in mean alveolar volume. DiNOS mice exhibited reduced amount of enlarged alveolar macrophages and altered their inflammatory phenotype relative to SP-D(−/−) mice. Neither disturbances of intracellular surfactant homeostasis nor the hyperplasia or hypertrophy of alveolar type II cells were corrected by iNOS ablation. However, despite a lack of effect on surfactant homeostasis, iNOS ablation did normalize lung function. The increased iNOS-activity in SP-D(−/−) mice is critical to the development of inflammation and consequent emphysema but is not important in altered surfactant homeostasis, implying that emphysema and alterations of type II cells are mediated via different pathways. The structural remodeling appears to dominate in terms of lung function.

Aspirin Therapy Enhances Lung Alveolarization in Newborn Pups Exposed to Neonatal Hyperoxia

Preterm infants with prolonged oxygen exposure are at risk of developing Bronchopulmonary Dysplasia (BPD). BPD is characterized by impairment of lung development and associated with inflammation. Aspirin is a cyclooxygenase inhibitor that disrupts pro-inflammatory pathways. We tested the hypothesis that aspirin therapy will improve lung function and growth following newborn hyperoxia exposure. Newborn mice were exposed to room air (RA) or >95% O2 and injected with vehicle (PBS) or 5 mg/kg aspirin (ASA) for 7 days. On day 7, O2 mice were returned to RA. Pulmonary function tests were performed on day 28 using the SCIREQ Flexivent system (n=9–11). Lungs were formalin fixed and paraffin embedded, and lung sections were H&E stained (n=3). Morphometric analysis was performed using Image Pro software. Total lung capacity (relative to body weight) was greater in PBS/O2 than PBS/RA and ASA/RA mice (78.20 ml/kg vs 50.49 ml/kg and 48.49 ml/kg, p<0.01). However, mice that received ASA during O2 exposure had lower total lung capacity (70.46 ml/kg, p<0.01). Alveolar number (per high power field) was greater in ASA/O2 mice than in the PBS/O2 mice (46 vs 69, p<0.05). Decreased total lung capacity was associated with an increase in alveolar number. Aspirin may be a beneficial therapy for preterm infants who are at risk of developing BPD and could improve long term lung development and growth.

INHALATION ADMINISTRATION OF ALL-TRANS-RETINOIC ACID FOR TREATMENT OF ELASTASE-INDUCED PULMONARY EMPHYSEMA IN FISCHER 344 RATS

A past study demonstrated that all-trans-retinoic acid (ATRA) treatment by intraperitoneal injection in a rat model of elastase-induced emphysema caused tissue regeneration as evidenced by a decrease in alveolar size and lung volume and an increase in alveolar number. We postulated that treatment with this retinoid by nose-only inhalation exposure would be a more efficient means of targeting damaged lung tissue. Emphysema was induced in male Fischer 344 rats by intratracheal instillation of pancreatic elastase (0.5 IU/g body weight). Four weeks after elastase instillation, animals were treated once daily, 4 days/week, for 3 weeks by exposing them nose-only to aerosolized ATRA (target concentration-time of 3000 or 15,000 mg-min/m3) or by injecting them intraperitoneally with ATRA in cottonseed oil (0.5 or 2.5 mg/kg). Based on estimates of particle deposition in the respiratory tract, inhalation doses were chosen to be consistent with injected doses. Lungs were fixed by inflation with formalin (constant pressure for 6 hours followed by >48 hours of immersion) and were embedded in paraffin. Sections were evaluated by histopathology and stereology. Inhalation exposure to ATRA at both aerosol concentrations caused significant elevations of ATRA in the lung, whereas only the high-dose injection treatment was associated with an elevation of lung ATRA. The mean ATRA concentration from lungs of rats in the high-dose inhalation exposure groups as measured by liquid chromatography–mass spectrometry was approximately 12-fold greater than that of high-dose injection-treated rats. Elastase instillation caused increased lung volumes, irregular alveolar air space enlargement, and fragmentation and attenuation of alveolar septa. Neither inhaled nor injected ATRA reduced the enlarged lung volumes associated with this emphysema model. Stereology demonstrated that alveolar air space enlargement in ATRA-treated rats was similar to that in sham-treated emphysematous animals. Thus, while inhalation treatment caused greater levels of the drug in lung tissue in comparison to that of injection-treated animals, treatment with ATRA by either route of administration did not cause a reversal of lung tissue damage in this model of elastase-induced emphysema.

Insulin-like growth factor-I gene expression in three models of accelerated lung growth

Background/Purpose : We have learned previously that in utero tracheal ligation reverses the structural and physiological effects of surgically created congenital diaphragmatic hernia. In addition, we have discovered that postnatal lung growth similarly can be accelerated using liquid-based airway distension with perfluorocarbon. Another model of accelerated lung growth is that of compensatory growth seen after neonatal pneumonectomy. In all of these models, growth has occurred because of an increase in alveolar number rather than enlargement of preexisting alveoli. However, the molecular mechanisms underlying these processes remain unknown. The purpose of this study was to determine if gene expression could be altered by changes in physical forces in the prenatal and postnatal lung. Methods : The three models of accelerated lung growth studied were the following: (1) The prenatal group, consisted of fetal lambs (n = 12) that underwent the surgical creation of a left diaphragmatic hernia at 90 days' gestation. Six of these animals also underwent simultaneous tracheal ligation. (2) The PFC group consisted of five neonatal animals that underwent isolation of the superior segment of the right upper lobe, with intrabronchial distension with perfluorocarbon to 7 to 10 mm Hg pressure for a 3-week period. (3) The postpneumonectomy group consisted of four neonatal animals that underwent left pneumonectomy. In the fetal study, lungs were retrieved at term (130 days), and in the postnatal study, lungs were retrieved 3 weeks after initial intervention. In all cases, RNA was extracted from snap-frozen lung samples and Northern blot analysis performed. Results : Insulinlike growth factor-I, insulinlike growth factor-II, and vascular endothelial growth factor gene expression were analyzed by densitometry. Insulinlike growth factor-I gene expression was found to be decreased in association with experimental diaphragmatic hernia ( P = .005), but restored to normal with tracheal ligation. Insulinlike growth factor-I gene expression was significantly increased in both postnatal models of accelerated lung growth ( P = .022, P = .016). No significant differences were found in insulinlike growth factor-II or vascular endothelial growth factor gene expression. Conclusions : The authors conclude from these preliminary data that (1) insulin like growth factor-I gene expression is reduced in experimental fetal diaphragmatic hernia and restored to normal by tracheal ligation, and (2) insulinlike growth factor-I gene expression is increased in both the liquid-based airway distension and postpneumonectomy models of accelerated postnatal lung growth. The authors speculate that all of these manipulations exploit a natural pathway essential for normal lung growth.

Alveolar development after ligation of left pulmonary artery in newborn pig: clinical relevance to unilateral pulmonary artery.

The left pulmonary artery and ductus arteriosus were ligated in 14 pigs at birth. Animals were killed at intervals between two and 24 weeks of age. In all animals, serial chest radiographs were taken. In eight animals, nitrogen-13 lung function studies showed normal fractional ventilation in both lungs and continued growth of the small left lung. After death, the fixed inflated volume was determined in each lung an alveolar development was analysed using quantitative morphometric techniques. In the right lung, after operation alveolar size increased considerably and at two weeks was similar to the normal at six months. The normal phase of rapid postnatal alveolar multiplication was delayed from the first to the second month of life. In all animals aged 20-24 weeks alveolar size was almost normal for age and an abnormal increase in alveolar number produced an abnormally large right lung. In the left lung, the alveoli multiplied to achieve a normal alveolar number in all animals sacrificed at 20 and 24 weeks, but the alveoli were abnormally small. Total lung volume was normal in all animals aged 20 and 24 weeks. The structural findings explained the radiographic and radionuclide investigations. The experimental findings are discussed in relation to lung growth in patients with an interrupted pulmonary artery.

1679 PHYSIOLOGIC SEQUELAE OF NO2-INDUCED BRONCHIOLITIS

To test the hypothesis that injury in the developing lung may lead to chronic obstructive lung disease, we studied the effects of NO2-induced bronchiolitis in newborn hamsters. Three day (d) Syrian hamsters were exposed continuously to 30-35 ppm NO2 23 hrs daily for 7 d. Pressure-volume and flow-volume curves during forced expiration at -50 cm H2O and minimal volumes (MV) were determined on excised lungs at 3 and 9 weeks (wk) of age. In NO2 animals studied at 3 wk (n=5), maximal expiratory flow at 40% total lung capacity (TLC) and elastic recoil pressure at 40% TLC were significantly lower than in control group (c) (n=4) (p<.02 and p<.04, respectively), while MV was significantly increased (p<.02). The upstream resistance (mean ± SEM) in the 3 wk NO2 group (.275 ± .058 cm H2O/ml/sec) was greater than in (c) (.209 ± .010) (p=NS). All measurements in 9 wk NO2 animals (n=4) were not significantly different from (c) (n=6). The decreased flow rates and increased MV in the NO2 group suggest small airways obstruction which may be related to decreased elastic recoil, increased airways resistance or both factors. The reversibility after 8 wk may result from 1) lung growth producing an increase in airways diameter, 2) alteration of the elastic properties of the lung associated with an increase in alveolar number during growth, or 3) physiologic accommodation to the effects of NO2. These data do not support the concept that acute lung injury is a progenitor of chronic lung disease. (Supp. by Amer. Lung Assn.)

New pathological findings in emphysema of childhood. 1. Polyalveolar lobe with emphysema.

A new pathological entity is here described—a polyalveolar lobe with or without emphysema—giving rise to the clinical features of childhood lobar emphysema. A detailed and quantitative study of the airways, alveoli and arteries was carried out on the left upper lobe removed because of shortness of breath, thought to be due to `childhood lobar emphysema9. The child was 17 days old and the radiograph showed hypertransradiancy of the left lung. The alveolar number was increased five-fold. Alveolar size was normal, so it was found that emphysema, accepted today as a structural diagnosis, was not present. The increase in alveolar number seemed confined to the apical, posterior, and anterior segments, the lingula being unaffected. By contrast, the airways and arteries were normal for age in number, size and structure, suggesting that the condition was a `giantism9 of the alveolar region. The blood flow was probably too low for the lobar volume; certainly the excessive alveolar number could not be due to increase in blood flow. In two further specimens, previously dissected, a similar polyalveolar condition was found, associated with emphysema. The patients were older at the time of surgical resection and the emphysema may have developed post-natally. In all three cases the radiographic features had suggested emphysema. It is suggested that the condition be called `polyalveolar lobe9, `with emphysema9 or `without emphysema9 being added as a separate item to the description.