Fatty diabetic lung: altered alveolar structure and surfactant protein expression.

Pulmonary dysfunction develops in type 2 diabetes mellitus (T2DM) in direct correlation with glycemia and is exacerbated by obesity; however, the associated structural derangement has not been quantified. We studied lungs from obese diabetic (fa/fa) male Zucker diabetic fatty (ZDF) rats at 4, 12, and 36 wk of age, before and after onset of T2DM, compared with lean nondiabetic (+/+) rats. Surfactant proteins A and C (SP-A and SP-C) immunoexpression in lung tissue was quantified at ages 14 and 18 wk, after the onset of T2DM. In fa/fa animals, lung volume was normal despite obesity. Numerous lipid droplets were visible within alveolar interstitium, lipofibroblasts, and macrophages, particularly in subpleural regions. Total triglyceride content was 136% higher. By 12 wk, septum volume was 21% higher, and alveolar duct volume was 36% lower. Capillary basement membrane was 29% thicker. Volume of lamellar bodies was 45% higher. By age 36 wk, volumes of interstitial collagen fibers, cells, and matrix were respectively 32, 25, and 80% higher, and capillary blood volume was 18% lower. ZDF rats exhibited a strain-specific increase in resistance of the air-blood diffusion barrier with age, which was exaggerated in fa/fa lungs compared with +/+ lungs. In fa/fa lungs, SP-A and SP-C expression were elevated at age 14-18 wk; the normal age-related increase in SP-A expression was accelerated, whereas SP-C expression declined with age. Thus lungs from obese T2DM animals develop many qualitatively similar changes as in type 1 diabetes mellitus but with extensive lipid deposition, altered alveolar type 2 cell ultrastructure, and surfactant protein expression patterns that suggest additive effects of hyperglycemia and lipotoxicity.

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex.

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.

Lack of response to all-trans retinoic acid supplementation in adult dogs following left pneumonectomy.

We showed previously that removing 55-58% of the lung by right pneumonectomy (R-PNX) in adult dogs triggers compensatory growth of the remaining lung, but removing 42-45% of the lung by left PNX (L-PNX) does not. We also showed that, following R-PNX, supplemental all-trans retinoic acid (RA) selectively enhances alveolar capillary endothelial cell volume (Yan X, Bellotto DJ, Foster DJ, Johnson RL, Jr., Hagler HH, Estrera AS, and Hsia CC. J Appl Physiol 96: 1080-1089, 2004). We hypothesized that RA supplementation might enhance compensation following L-PNX and tested this hypothesis by administering RA (2 mg.kg(-1).day(-1), 4 days/wk) or placebo orally to litter-matched adult foxhounds for 4 mo following L-PNX. Resting lung function was measured under anesthesia. Air and tissue volumes of the remaining lung were assessed by high-resolution computed tomography scan and by detailed postmortem morphometric analysis of the fixed lung. There was no significant difference in resting lung function, lung volume, alveolar structure, or septal ultrastructure between RA and placebo treatment groups. We conclude that RA supplementation does not induce post-PNX compensatory lung growth in the absence of existing cellular growth activities initiated by other primary signals.

Enhanced alveolar growth and remodeling in Guinea pigs raised at high altitude.

To examine the effects of chronic high altitude (HA) exposure on lung structure during somatic maturation, we raised male weanling guinea pigs at HA (3800m) for 1, 3, or 6 months, while their respective male littermates were simultaneously raised at low altitude (LA, 1200m). Under anaesthesia, airway pressure was measured at different lung volumes. The right lung was fixed at a constant airway pressure for morphometric analysis under light and electron microscopy. In animals raised at HA for 1 month, lung volume, alveolar surface area and alveolar-capillary blood volume (V(c)) were elevated above LA control values. Following 3-6 months of HA exposure, increases in lung volume and alveolar surface area persisted while the initial increase in V(c) normalized. Additional adaptation occurred, including a higher epithelial cell volume, septal tissue volume and capillary surface area, a lower alveolar duct volume and lower harmonic mean diffusion barrier resulting in higher membrane and lung diffusing capacities. These data demonstrate enhanced alveolar septal growth and progressive acinar remodeling during chronic HA exposure with long-term augmentation of alveolar dimensions as well as functional compensation in lung compliance and diffusive gas transport.

Persistent structural adaptation in the lungs of guinea pigs raised at high altitude.

Laboratory guinea pigs raised at high altitude (HA, 3800 m) for up to 6 mo exhibit enhanced alveolar growth and remodeling (Hsia et al., 2005. Resp. Physiol. Neurobiol. 147, 105-115). To determine whether initial HA-induced structural enhancement persists following return to intermediate altitude (IA), we raised weanling guinea pigs at (a) HA for 11-12 mo, (b) IA (1200 m) for 11-12 mo, and (c) HA for 4 mo followed by IA for 7-8 mo (HA-to-IA). Morphometric analysis was performed under light and electron microscopy. Body weight and lung volume were similar among groups. Prolonged HA residence increased alveolar epithelium and interstitium volumes while reducing alveolar-capillary blood volume. The HA-induced gains in type-1 epithelium volume and alveolar surface area were no longer present following return to IA whereas volume increases in type-2 epithelium and interstitium and the reduction in alveolar duct volume persisted. Results demonstrate persistent augmentation of some but not all aspects of lung structure throughout prolonged HA residence, with partial reversibility following re-acclimatization to IA.

Retinoic acid induces nonuniform alveolar septal growth after right pneumonectomy.

To determine whether all-trans retinoic acid (RA) enhances compensatory lung growth in fully mature animals, adult male dogs (n = 4) received 2 mg x kg(-1) x day(-1) po RA 4 days/wk beginning the day after right pneumonectomy (R-PNX, 55-58% resection). Litter-matched male R-PNX controls (n = 4) received placebo. After 4 mo, the remaining lung was fixed by tracheal instillation of fixatives at a constant airway pressure for detailed morphometric analysis. After RA treatment compared with placebo, lung volume was slightly but not significantly lower. Volume density of septum to lung was 37% higher because of a 50 and 25% higher volume density of capillary and septal tissue, respectively. Mean septal thickness was 27% higher. Absolute volumes of endothelial cells and capillary blood were 31-37% higher, whereas epithelial and interstitial volumes were not different between groups. Absolute alveolar-capillary surface areas did not differ between groups, and alveolar septal surface-to-volume ratio was 20% lower in RA-treated animals. RA treatment exaggerated interlobar differences in morphometric indexes and caused alveolar capillary morphology to revert to a more immature state. Thus RA treatment during early post-R-PNX adaptation preferentially enhanced alveolar capillary and endothelial cell volumes consistent with formation of new capillaries, but the associated septal distortion precluded a corresponding increase in gas-exchange surface or morphometric estimates of lung diffusing capacity.

Effect of ophthalmic viscosurgical devices on lens epithelial cells: a morphological study.

PURPOSE: To investigate the morphological effects of Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%) on lens epithelial cells (LECs). SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA, and the Laboratory of Ultrastructural Morphology, Zoological Institute, University of Liège, Liège, Belgium. METHODS: Human LECs collected via capsulorhexis were examined by light microscopy (LM) and transmission electron microscopy (TEM). Lens epithelial cells from rabbit capsulorhexis samples were studied by LM and TEM following exposure to Provisc (sodium hyaluronate 1.0%) or Viscoat ophthalmic viscoelastic device (OVD). Since Viscoat is hypertonic (340 mOsm), hypertonic, isotonic, and hypotonic solutions were compared to investigate a possible mechanism for the observed effects. The effects of Provisc and Viscoat on rabbit LECs in the intact lens were also compared. RESULTS: Human LECs gathered via capsulorhexis following exposure to Viscoat were generally thinner than control samples and often had condensed nuclei and increased intracellular vacuolization. Rabbit capsular tissue exposed in situ to Viscoat demonstrated changes similar to those seen in humans. Cells exposed to Provisc were similar to cells in untreated controls in humans and rabbits. Corneal endothelial cells exposed to either agent were unaffected. Experiments with hypertonic and hypotonic buffers induced some of the changes noted with Viscoat, but the effects were less severe. Lens epithelial cells in intact rabbit lenses exposed to Viscoat appeared similar to LECs in the control samples. CONCLUSIONS: Light microscopy and TEM of human lens capsule tissue suggest that Viscoat induces significant morphological changes in LECs during cataract surgery. The changes may underlie the improved visualization of these cells that has been reported during cataract surgery. Corneal endothelial cells were unaffected by exposure to Viscoat. Studies in a rabbit model suggest that the hyperosmolarity of Viscoat may play a partial role in the LEC changes.

Defining a stimuli-response relationship in compensatory lung growth following major resection.

Major lung resection is a robust model that mimics the consequences of loss-of-functioning lung units. We previously observed in adult canines, following 42% and 58% lung resection, a critical threshold of stimuli intensity for the initiation of compensatory lung growth. To define the range and limits of this stimuli-response relationship, we performed morphometric analysis on the remaining lobes of adult dogs, 2-3 years after surgical removal of ∼ 70% of lung units in the presence or absence of mediastinal shift. Results were expressed as ratios to that in corresponding control lobes. Lobar expansion and extravascular tissue growth (∼ 3.8- and ∼ 2.0-fold of normal, respectively) were heterogeneous; the lobes remaining next to the diaphragm exhibited a greater response. Tissue growth and capillary formation, indexed by double-capillary profiles, increased, regardless of mediastinal shift. Septal collagen fibers increased up to 2.7-fold, suggesting a greater need for structural support. Compared with previous cohorts following less-extensive resection, tissue volume and gas-exchange surface areas increased significantly only in the infracardiac lobe following 42% resection, exceeded two- to threefold in all lobes following 58% resection, and then exhibited diminished gains following ∼ 70% resection. In contrast, alveolar-capillary formation increased with incremental resection without reaching an upper limit. Overall structural regrowth was most vigorous and uniform following 58% resection. The diminishment of gains in tissue growth, following ∼ 70% resection, could reflect excessive or maldistributed mechanical stress that threatens septal integrity. Results also suggest additional independent stimuli of alveolar-capillary formation, possibly related to the postresection augmentation of regional perfusion.

Separating in vivo mechanical stimuli for postpneumonectomy compensation: imaging and ultrastructural assessment.

Following right pneumonectomy (PNX), the remaining lung expands and its perfusion more than doubles. Tissue and microvascular mechanical stresses are putative stimuli for compensatory lung growth and remodeling, but their relative contribution remains uncertain. To temporally separate expansion- and perfusion-related stimuli, we replaced the right lung of adult dogs with a customized inflated prosthesis. Four months later, the prosthesis was either acutely deflated (DEF) or kept inflated (INF). Thoracic high-resolution computed tomography (HRCT) was performed pre- and post-PNX before and after prosthesis deflation. Lungs were fixed for morphometric analysis ∼12 mo post-PNX. The INF prosthesis prevented mediastinal shift and lateral lung expansion while allowing the remaining lung to expand 27-38% via caudal elongation, associated with reversible capillary congestion in dependent regions at low inflation and 40-60% increases in the volumes of alveolar sepal cells, matrix, and fibers. Delayed prosthesis deflation led to further significant increases in lung volume, alveolar tissue volumes, and alveolar-capillary surface areas. At postmortem, alveolar tissue volumes were 33% higher in the DEF than the INF group. Lateral expansion explains ∼65% of the total post-PNX increase in left lung volume assessed in vivo or ex vivo, ∼36% of the increase in HRCT-derived (tissue + microvascular blood) volume, ∼45% of the increase in ex vivo septal extravascular tissue volume, and 60% of the increase in gas exchange surface areas. This partition agrees with independent physiological measurements obtained in these animals. We conclude that in vivo signals related to lung expansion and perfusion contribute separately and nearly equally to post-PNX growth and remodeling.

Fatty diabetic lung: functional impairment in a model of metabolic syndrome.

The Zucker diabetic fatty (ZDF fa/fa) rat with genetic leptin insensitivity develops obesity and Type 2 diabetes mellitus (T2DM) with age accompanied by hyperplastic changes in the distal lung (Am J Physiol Lung Cell Mol Physiol 298: L392-L403, 2010). To determine the functional consequences of structural changes, we developed a rebreathing (RB) technique to simultaneously measure lung volume, pulmonary blood flow, lung diffusing capacity (Dl(CO)), membrane diffusing capacity (Dm(CO)), pulmonary capillary blood volume (Vc), and septal tissue volume in anesthetized tracheostomized male ZDF fa/fa and matched lean (+/+) control animals at 4, 8, and 12 mo of age. Results obtained by RB technique were compared with that measured by a single-breath (SB) technique and to that expected in a wide range of species. In fa/fa animals compared with +/+, lung volumes and compliance were 13-35% lower at different ages, and the normal age-related increase in lung compliance was no longer evident. Mean pulmonary blood flow declined with age in fa/fa but not in +/+ animals. Dl(CO) measured at a given pulmonary blood flow was 20-43% lower at different ages due to reductions in both Dm(CO) and Vc. Septal tissue volume was also reduced in older fa/fa rats. We conclude that obese rats with T2DM develop significant restrictive pulmonary defects with diffusion impairment in a pattern similar to that previously reported in obese human subjects with T2DM. Functional impairment became exaggerated with age and duration of T2DM. In both fa/fa and +/+ animals, Dl(CO) measured by RB was systematically higher than by SB technique whereas lung volume was similar, a finding consistent with heterogeneous distribution of ventilation in the rat lung.

Permanent alveolar remodeling in canine lung induced by high-altitude residence during maturation.

Young canines born at sea level (SL) and raised for 5 mo at high altitude (HA, 3,800 m), followed by return to SL before somatic maturation, showed enhanced alveolar gas exchange and diffusing capacity at rest and exercise that persisted into adulthood (McDonough P, Dane DM, Hsia CC, Yilmaz C, Johnson RL Jr. J Appl Physiol 100: 474-81, 2006; Hsia CCW, Johnson RL Jr, McDonough P, Dane DM, Hurst MD, Fehmel JL, Wagner HE, Wagner PD. J Appl Physiol 102: 1448-55, 2007). To examine the associated structural response, we quantified lung ultrastructure in male foxhounds raised at 3,800 m HA or their littermates raised at SL (n = 6 each) from 2.5 to 7.5 mo of age. Three years following return to SL, lungs were fixed for morphometric analysis. In HA-exposed animals compared with SL controls, lung volume at a given inflation pressure was higher with enlargement of alveolar ducts and sacs without significant differences in the volumes of alveolar cell components, septal tissue, or in alveolar-capillary surface areas. There was a shift toward a significantly lower harmonic mean thickness of the blood-gas diffusion barrier in HA-raised animals. As a control organ, muscle capillary length density of costal diaphragm was significantly higher in HA-raised animals, indicating parallel adaptation in oxygen transport organs. We conclude that, in actively growing animals, 5 mo of HA exposure that was discontinued before somatic maturation induced acinar remodeling that increased lung compliance and reduced the resistance of blood-gas diffusion barrier to diffusion that persisted into adulthood, but without permanent enhancement of alveolar tissue growth.

Noninvasive quantification of heterogeneous lung growth following extensive lung resection by high-resolution computed tomography.

To quantify the in vivo magnitude and distribution of regional compensatory lung growth following extensive lung resection, we performed high-resolution computed tomography at 15- and 30-cmH(2)O transpulmonary pressures and measured air and tissue (including microvascular blood) volumes within and among lobes in six adult male foxhounds, before and after balanced 65% lung resection ( approximately 32% removed from each side). Each lobe was identified from lobar fissures. Intralobar gradients in air and tissue volumes were expressed along standardized x,y,z-coordinate axes. Fractional tissue volume (FTV) was calculated as the volume ratio of tissue/(tissue + air). Following resection compared with before, lobar air and tissue volumes increased 1.8- to 3.5-fold, and whole lung air and tissue volumes were 67 and 90% of normal, respectively. Lobar-specific compliance doubled post-resection, and whole lung-specific compliance normalized. These results are consistent with vigorous compensatory growth in all remaining lobes. Compared with pre-resection, post-resection interlobar heterogeneity of FTV, assessed from the coefficient of variation, decreased at submaximal inflation, but was unchanged at maximal inflation. The coefficient of variation of intralobar FTV gradients changed variably due to the patchy development of thickened pleura and alveolar septa, with elevated alveolar septal density and connective tissue content in posterior-caudal and peripheral regions of the remaining lobes; these areas likely experienced disproportional mechanical stress. We conclude that HRCT can noninvasively and quantitatively assess the magnitude and spatial distribution of compensatory lung growth. Following extensive resection, heterogeneous regional mechanical lung strain may exceed the level that could be sustained solely by existing connective tissue elements.

Postpneumonectomy lung expansion elicits hypoxia-inducible factor-1alpha signaling.

We (42) previously reported differential regulation of hypoxia-inducible factors (HIF-1alpha, -2alpha, and -3alpha) mRNA in canine lungs during normal maturation and postpneumonectomy (PNX) compensatory growth in the absence of overt hypoxia. To test the hypothesis that lung expansion activates HIF signaling, we replaced the right lung of six adult foxhounds with inflated custom-shaped silicone prosthesis to keep the mediastinum in the midline and minimize lateral expansion of the remaining lung. After 3 wk of recovery and stabilization of perfusion, the prosthesis was acutely deflated in three animals, causing the remaining lung to expand by 114%. In three other animals, the prosthesis remained inflated. Three days following deflation, we observed significant elevation in the mRNA and nuclear protein levels of HIF-1alpha ( approximately 60%) as well as activation of its transcriptional regulator, the serine/threonine protein kinase B (phospho-Akt-to-total Akt ratio, 124%), and the mRNA and protein levels of its downstream targets, erythropoietin receptor (71-183%) as well as VEGF (33-58%) compared with the pre-PNX control lung from the same animal. The mRNA of HIF-2alpha, HIF-3alpha, and VEGF receptors did not change with acute deflation. We conclude that in vivo lung expansion by post-PNX deflation of space-occupying prosthesis elicits coordinated activation of HIF-1alpha signaling in adult lungs. This pathway could play an important role in mediating lung growth and remodeling during maturation and post-PNX compensation.

Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth.

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.