[Pulmonary tissue regeneration -- a hope for the future].
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Animals↗
Biomedical subjects
H Fehrenbach
Publications and source records attributed to H Fehrenbach.
[Pathogenesis of chronic obstructive pulmonary disease].
It is currently believed that the most important factor in the pathogenesis of chronic obstructive pulmonary disease (COPD) is inflammation of the small airways caused by inhaled particles and gases. In this context, a disturbance of the physiological balance between proteases and antiproteases develops that may cause lung emphysema. Moreover, oxidative stress seems to be important, as it may enhance the inflammatory reaction. The development of emphysema may also involve a loss of alveolar cells by apoptosis. Finally, several studies have indicated that a systemic inflammation is induced by COPD that may be of relevance to the development of systemic components that are observed in COPD patients.
Air Pollutants↗
Involvement of distal airways in a chronic model of experimental asthma.
BACKGROUND: Bronchial asthma is characterized by chronic airway inflammation and airway remodelling which occurs in both proximal and distal airways. These changes are associated with development of airway hyper-responsiveness and airflow limitation. OBJECTIVE: This study was aimed to analyse whether chronic inhalative allergen challenges in mice lead to morphological and physiological changes comparable with this phenotype. METHODS: For this purpose, BALB/c mice were systemically sensitized to ovalbumin (OVA) followed by aerosol allergen challenges on 2 consecutive days per week for 12 weeks. RESULTS: In chronically challenged mice, tissue inflammation in proximal as well as distal airways was observed with a predominance of lymphocytes within the cellular infiltrate. In contrast, inflammation in the airway lumen decreased over time. These changes were associated by a shift in bronchoalveolar lavage-cytokine levels from IL-4, IL-5 and TNF-alpha production (during the acute phase) towards markedly increased levels of TGF-beta during the chronic phase. Goblet cell hyperplasia and subepithelial fibrosis occurred throughout the airway tree. In terms of lung function, chronically challenged mice developed persistent bronchial hyper-responsiveness and progressive airflow limitation. Six weeks after OVA aerosol discontinuation, airway inflammation still persisted although lung function was normalized. CONCLUSION: These data indicate that our model of chronic aerosol allergen challenges leads to a phenotype of experimental asthma with participation of distal airways and persistence of inflammation thereby resembling many morphological and physiological aspects of human bronchial asthma.
Administration, Inhalation↗
Alveolization: does "A" stand for appropriate morphometry?
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Animals↗
Characterisation of post-pneumonectomy lung growth in adult mice.
A model of inducible expansion of the gas exchange area in adult mice would be ideal for the investigation of molecular determinants of airspace regeneration in vivo. Therefore, the post-pneumonectomy (post-PNX) compensatory lung growth in adult C57BL/6 mice was characterised in this study. Mice underwent left-sided PNX. Right lung volume was assessed on days 1, 3, 5, 7, 10 and 21 after PNX, and total DNA and cellular proliferation of the right lung were determined. Lung histology was studied using immunohistochemistry and quantitatively characterised by detailed stereological investigations. Pulmonary function was assessed using a mouse body-plethysmograph. Following PNX, right-lung volume rapidly restored the initial volume of left and right lung. Total DNA increased significantly over 21 days and equalled the total DNA amount of both lungs in the control mice. Septal cell proliferation significantly increased after PNX, and included endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. Stereological investigations of left and right control lungs versus right lungs 21 days after PNX indicated complete restoration of body mass-specific alveolar surface area. Pulmonary function testing showed marked alteration at 3 days and normalisation at 21 days post-PNX. In conclusion, well reproducible reconstitution of alveolar gas-exchange surface based on septal tissue expansion may be provoked by pneumonectomy in adult mice.
Animals↗
[Aerosols in medicine and veterinary medicine].
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Aerosols↗
Alveolar macrophages are the main source for tumour necrosis factor-alpha in patients with sarcoidosis.
Tumour necrosis factor (TNF)-alpha is known to play a major role in the formation of noncaseating granuloma, a hallmark of sarcoidosis. The main cellular source in situ is still ambiguous. Serial sections of transbronchial biopsies from 14 patients with and 12 without sarcoidosis were studied, using immunohistochemistry (IHC), for TNF-alpha, T-cells (CD3), macrophages (CD68), and epithelial cells (MNF116). TNF-alpha spontaneously released (sr-TNF-alpha) by freshly isolated bronchoalveolar lavage cells, isolated from the same patients and cultured without any stimulus over a 24-h period was measured using an enzyme-linked immunosorbent assay. IHC revealed colocalisation of TNF-alpha with CD68 cells only. Cases with TNF-alpha tissue immunoreactivity exhibited higher sr-TNF-alpha (1,667 +/- 504 pg x mL(-1)) than cases without tissue immunoreactivity (211 +/- 60 pg x mL(-1)). In an explorative approach, a subgroup of patients could be identified and characterised by the presence of alveolar macrophage aggregates. It was found that sr-TNF-alpha was highest in this subgroup (2,700 +/- 769 pg x mL(-1)) compared with patients with normal histology (221 +/- 61 pg x mL(-1)) or with prominent granuloma (460 +/- 137 pg x mL(-1)), whereas in most clinical parameters this subgroup was intermediate. The findings from this study strongly corroborate the view that alveolar macrophages are the main cellular source for tumour necrosis factor-alpha in the initial phase of sarcoidosis. The authors suggest that in these patients, aggregates of alveolar macrophages may represent at least predecessors to granulomas if not granulomas in statu nascendi.
Adult↗
Increased surfactant protein D in rat airway goblet and Clara cells during ovalbumin-induced allergic airway inflammation.
BACKGROUND: Structural remodelling of airways in asthma that follows inflammation may be affected by surfactant protein D (SP-D)-mediated effects on the immune response. OBJECTIVE: To determine potential sites of SP-D interaction with the pulmonary immune response, we examined the distribution of immunoreactive SP-D in an experimental model of allergen-induced airway inflammation using immunohistochemistry, biochemical methods and in situ hybridization. METHODS: The experimental model used subcutaneous injection of ovalbumin in adult rats, which induced an airway response to inhaled nebulized ovalbumin. Three groups of rats (ovalbumin, ovalbumin + dexamethasone and saline) were challenged thrice weekly for 3 weeks. A fourth group of seven rats (naive) were taken from the same delivery of rats as the other groups. Lungs were then lavaged to determine total cell count, eosinophil count, ovalbumin-specific IgE by enzyme-linked immunosorbent assay and SP-D by immunoblot. Tissue samples were fixed and embedded, and sections were studied for the infiltration of eosinophils and for expression of SP-D protein by histochemistry and mRNA by in situ hybridization. RESULTS: Ovalbumin induced perivascular and peribronchiolar eosinophilia which could be prevented by dexamethasone treatment. In addition, the ovalbumin-specific IgE levels in serum and bronchoalveolar lavage fluid of ovalbumin-challenged animals were enhanced. Increased amount of SP-D in lavage and tissue, particularly in type II pneumocytes, in Clara cells and, surprisingly, in hyperplastic goblet cells of inflamed lungs was found. SP-D mRNA was detected in goblet cells as well as in type II pneumocytes and Clara cells. Dexamethasone treatment did not affect level of SP-D immunoreactivity. CONCLUSION: SP-D accumulation is increased in this model of allergen-induced eosinophilia, both in upper and lower airways. The increase is unaffected by dexamethasone.
Analysis of Variance↗
Keratinocyte growth factor-induced proliferation of rat airway epithelium is restricted to Clara cells in vivo.
Keratinocyte growth factor (KGF) is a potent mitogen of pulmonary bronchial and alveolar epithelial cells. However, it is unclear which type(s) of airway epithelial cells (AEC) proliferate(s) in response to KGF. AEC proliferation was induced in rats by either endobronchial instillation of 5 mg recombinant human (rHu) KGF per kg body weight or by adenoviral transfer of the human KGF gene (Ad5-HuKGF). Alterations in terminal airway AEC were followed for up to 7 days after rHuKGF, and for up to 28 days after Ad5-HuKGF. Cell proliferation, as assessed by immunohistochemistry (IHC) for incorporated 5-bromo-2'-deoxyuridine (BrdU) and quantified by stereology, peaked at days 1-2 and was resolved by day 7 after rHuKGF and by day 21 after Ad5-HuKGF. Double immunofluorescence labelling for BrdU or Ki-67 on the one hand, and for Clara cell specific protein 10 (CC10) and calcitonin-gene related peptide on the other hand, demonstrated that Clara cells, not pulmonary neuroendocrine cells, proliferated in response to human KGF. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) method in conjunction with IHC for MNF116 failed to detect significant numbers of apoptotic AEC. IHC in conjunction with stereology revealed transient phenotypic alterations with a decrease in CC10, an increase in surfactant protein D and an increase in CD44v6 in AEC. The authors conclude that Clara cells responded to human keratinocyte growth factor in vivo by proliferation as well as by changes in protein expression, whereas no significant response was observed in pulmonary neuroendocrine cells. As Clara cells are intimately involved in airway epithelial repair, ion and fluid transport, and modulate lung inflammation, the potential of human keratinocyte growth factor to protect the lung may in part rely on the response of Clara cells.
Animals↗
Ultrastructure of canine type II pneumocytes during hypothermic ischemia of the lung: a study by means of conventional and energy filtering transmission electron microscopy and stereology.
Alterations in pulmonary surfactant have been reported to be associated with ischemia/reperfusion injury in experimental and clinical lung transplantation. It is unknown whether these alterations are due to damage to surfactant synthesizing type II pneumocytes during hypothermic ischemic storage. The aim of the present study was to examine the effects of hypothermic ischemic storage of the lung on canine type II pneumocytes by means of conventional (CTEM) and energy filtering TEM (EFTEM) and stereology. The lungs of 18 dogs were fixed for TEM immediately after cardiac arrest (6 double lungs) and after storage in Tutofusin at 4 degrees C for 20 min, 4 hr, 8 hr, and 12 hr (6 single lungs, respectively). Using a systematic uniform random sampling scheme, type II pneumocytes were analyzed qualitatively and stereologically. The relative phosphorus content of cell organelles, especially the surfactant containing lamellar bodies, was investigated by EFTEM. By CTEM, no major qualitative alterations could be observed in type II pneumocytes of the experimental groups. Stereologically, no significant changes in the volume densities or the volume-to-surface ratios of type II pneumocytes and their lamellar bodies were found. By EFTEM, the highest intracellular phosphorus signals were recorded over lamellar bodies in all experimental groups. No changes in the phosphorus signals were observed during ischemia. These results indicate that the ultrastructure of canine type II pneumocytes and their lamellar bodies is not affected by hypothermic ischemia of the lung up to 12 hr. Structural preservation of intracellular surfactant is possible during prolonged ischemic lung storage.
Animals↗
Alveolar epithelial type II cell: defender of the alveolus revisited.
In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2) cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today.
Animals↗
Nitroglycerin alters alveolar type II cell ultrastructure after ischemia and reperfusion.
BACKGROUND: Although administration of nitric oxide (NO) has been suggested to reduce pulmonary reimplantation response, concerns remain about cytotoxic side effects. METHODS: Using light and electron microscopy, we examined the effects of the NO donor nitroglycerin (NTG) (0.1 mg/ml) as a supplement to the preservation solution Celsior on the structural integrity of rat lungs after extracorporeal ischemia (4 hours at 10 degrees C) and reperfusion (50 minutes) (IR). We performed evaluation in comparison with Celsior alone after IR using either standard antegrade perfusion through the pulmonary artery or retrograde perfusion through the left atrium as an alternative way to improve the preservation quality. Untreated, non-ischemic lungs served as controls (n = 5 per group). We recorded respiratory and hemodynamic parameters during reperfusion. Tissue collection using systematic uniform random sampling was representative for the whole organ and allowed stereologic quantification of structures. RESULTS: After IR, histochemistry revealed no breaks in the alveolo-capillary barrier and we detected no alveolar flooding. Edema formed in the peribronchovascular cuffs, of which the volume fraction was increased (p =.008). Vasoconstriction of the smaller arteries accompanied antegrade flush, which occurred neither after administration of NTG nor after retrograde flush, as shown by immunostaining for alpha-smooth muscle actin. Treatment with NTG was associated with focal disintegration of Type II cells, which displayed edematous swelling of distinct cell compartments and lysis of mitochondria and cells. Nitroglycerin prevented alveolar collapse, which was increased in the other IR groups (p = 0.013). We observed alterations in intra-alveolar surfactant components. CONCLUSION: These findings indicate pathologic effects of NTG treatment on alveolar epithelial integrity. Therefore, we suggest further critical evaluation of NTG/NO for therapeutic use in lung transplantation.
Animals↗
Up-regulated expression of the receptor for advanced glycation end products in cultured rat hepatic stellate cells during transdifferentiation to myofibroblasts.
Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell-surface molecules. Blockade of RAGE has been reported to considerably improve liver function and accelerate regeneration after hepatectomy. The aim of this study was to investigate the cell type-specific expression of RAGE, and to examine whether transdifferentiation of hepatic stellate cells (HSC) into myofibroblasts (MFB) is associated with changes in RAGE expression. Northern blot analysis revealed that RAGE mRNA was exclusively expressed by HSC isolated from rat liver, while no transcripts were seen in hepatocytes, Kupffer cells, or sinusoidal endothelial cells. Expression of RAGE mRNA was up-regulated during transdifferentiation of HSC into MFB. Concomitantly, expression of RAGE protein was increased as confirmed by Western blotting and immunohistochemistry. As assessed by radioactive labeling, transforming growth factor beta(1) (TGF-beta(1)) induced a time-dependent 2- to 15-fold increase in the de novo synthesis of RAGE protein, which was completely abolished using PD098059, a specific inhibitor of the mitogen-activated protein kinase (MAPK) kinase. As shown by double-immunofluorescence staining, RAGE colocalized with alpha-smooth muscle actin, and immunoelectron microscopy demonstrated the most prominent labeling for RAGE at filopodial membranes of MFB. In conclusion, this study demonstrates that expression of RAGE is restricted to rat HSC, and that expression is up-regulated during activation of HSC and transition to MFB. The preferential immunogold labeling of RAGE to focal membrane areas of filopodia of MFB is suggestive of a role of RAGE in the spreading and migration of activated HSC/MFB, major players in liver fibrogenesis.
Animals↗
Changes in xylosyltransferase activity and in proteoglycan deposition in bleomycin-induced lung injury in rat.
Several lines of evidence support the hypothesis of the involvement of altered proteoglycan deposition in the development of lung diseases. UDP-D-xylose: core protein beta-D-xylosyltransferase (UDP-xylosyltransferase; EC 2.4.2.26) is a key enzyme for the glycosylation of proteoglycan core proteins. This study examined the catalytic activity of UDP-xylosyltransferase in lung tissue and in isolated fibroblasts, as well as the deposition of the proteoglycans versican, biglycan and decorin in rat lung tissue during bleomycin-induced lung injury. Rats were given, endotracheally, a single dose of bleomycin. Deposition of proteoglycans in lung tissue was assessed by immunohistochemistry and the catalytic activity of xylosyltransferase was determined with an acceptor peptide of the sequence Q-E-E-E-G-S-G-G-G-Q-G-G as a substrate. The results show coincidence of increasing xylosyltransferase activities in lung tissue with accumulation of versican at alveolar entrance rings and in fibrotic regions in close proximity to alpha-smooth muscle actin-positive cells. In contrast, no changes in biglycan and decorin deposition in fibrotic lungs were observed, except for decorin in alveolar type II pneumocytes and alveolar macrophages. Bleomycin treatment of isolated rat lung fibroblasts resulted in a concentration-dependent increase of xylosyltransferase activity up to 2 mU bleomycin x mL(-1). The data suggest a participation of myofibroblasts with increased xylosyltransferase activities in accumulation of versican in fibrotic foci of injured lung tissue at the early stages of development of lung fibrosis.
Animals↗
Alveolar epithelial type II cell apoptosis in vivo during resolution of keratinocyte growth factor-induced hyperplasia in the rat.
Keratinocyte growth factor (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF/kg body weight or diluent. Lungs were fixed 1, 2, 3, 5, and 7 days later. Apoptosis was identified by TdT-mediated dUTP nick-end labeling (TUNEL), double-labeling for TUNEL and the type II cell marker MNF116, and electron microscopy. Fas, FasL, Bax, Bcl-2, and pro- and active caspase-3 were studied by immunohistochemistry. Changes were quantified by stereology. Cell type specificity was investigated by immunofluorescence double staining. Type II cells exhibited Fas, FasL, Bcl-2, and procaspase-3 irrespective of treatment and time. Immunoelectron microscopy revealed Fas at the apical type II cell membrane. Bax staining was prominent in controls (45-95% of type II cell surface fraction), markedly decreased during hyperplasia at days 2 (20-40%) and 3 (0-10%), and reappeared at day 7 (25-45%) when apoptosis was prominent. Remnants of apoptotic type II cells were incorporated in membrane-bound vacuoles of type II cell neighbors as well as alveolar macrophages. The results indicate that type II cells can enter the Fas/FasL/caspase-3 pathway regulated by Bax and Bcl-2. High Bcl-2:Bax levels favor type II cell survival and a low rate of apoptosis during hyperplasia. Low Bcl-2:Bax levels favor type II cell apoptosis during resolution. Because of time-dependent changes that occur within a short time, the KGF-treated rat lung provides a useful in vivo model to investigate apoptosis in the context of tissue remodeling and repair.
Animals↗
Resistance of L132 lung cell clusters to glyoxal-induced apoptosis.
Glyoxal is a highly reactive glycating agent involved in the formation of advanced glycation end products (AGEs) and known to induce apoptosis. AGE-mediated apoptosis may be an important mechanism of alveolar epithelial remodelling in pulmonary fibrosis. In this study, we investigated the cytotoxic effect of glyoxal on the fetal human epithelial lung cell line L132 under serum-free conditions. This type of culture, which forces the cells to grow as spheroids, also excludes effects of preformed AGEs by the reaction of glyoxal with fetal calf serum proteins. Our results showed that in cells treated with 200 microM glyoxal, the intercellular contacts in spheroids were disrupted, i.e. cells became totally dissociated. Immunocytochemical analysis revealed a dose-dependent accumulation of the AGE product epsilonN-(carboxymethyl)lysine (CML) in cells detached from cell clusters. The loss of cell attachment was associated with decreased expression of beta1-integrins and CD44 as revealed by laser scanning cytometry (LSC). Increasing concentrations of glyoxal induced an increase in the number of apoptotic cells which were identified by the immunoreactivity for active caspase-3. Remaining cell clusters showed resistance to both CML formation and apoptosis. The present findings demonstrate that cells treated with glyoxal undergo possibly anoikis, a specific mode of apoptosis caused by loss of cell adhesion.
Anoikis↗
Preservation of intraalveolar surfactant in a rat lung ischaemia/reperfusion injury model.
Ischaemia/reperfusion (I/R) injury, a major problem in clinical lung transplantation, is associated with surfactant dysfunction. The present study aimed to test the hypothesis that preservation related improvements in post-ischaemic lung function are associated with improved ultrastructural preservation of pulmonary surfactant. Rat lungs were flush perfused with modified Euro-Collins solutions (ECS), stored for 2 h at 4 degrees C, and reperfused for 40 min. Lungs were preserved with conventional (ECS 115: 115 mmol x L(-1) K+), medium-K+ (ECS 40: 40 mmol x L(-1) K+), or low-K+ (ECS 10: 10 mmol x L(-1) K+) ECS. Functional parameters were monitored during reperfusion (n=10 per group). After reperfusion, left lungs were prepared for electron microscopical and stereological analysis of surfactant (n=5 per group). In all three experimental groups notable I/R injury developed which was lowest in ECS 40 as indicated by significantly less intraalveolar oedema, higher perfusate oxygenation, and lower peak inspiratory pressure. This was associated with a significantly superior preservation of the ultrastructure of the surface active surfactant subtype tubular myelin in ECS 40 compared with ECS 115 and ECS 10. Stereological analysis revealed that the relative amount of tubular myelin was highest in ECS 40 (mean+/-SEM; 6.2+/-0.8%) compared with ECS 115 (3.0+/-1.0%) and ECS 10 (2.7+/-1.6%). Analysis of surfactant in its natural location within the organ showed that the severity of ischaemia/reperfusion injury correlates with differences in intraalveolar surfactant composition. Improved post-ischaemic respiratory function achieved by medium-K+ Euro-Collins solution is associated with superior ultrastructural preservation of tubular myelin. It is concluded that the integrity of surface active tubular myelin represents an important criterion for the assessment of lung preservation quality.
Animals↗
Immunohistochemical evidence for the occurrence of similar epithelial phenotypes during lung development and radiation-induced fibrogenesis.
PURPOSE: Processes of fibrosis, wound healing and tissue regeneration have in common the fact that proliferation and differentiation of cells involved in the restoration of normal-tissue architecture resemble to a certain degree the embryonic development of the corresponding tissue. The present review focuses on the phenotypic changes of alveolar epithelial cells during fibrogenesis and describes similarities in the expression pattern of epithelial antigens during lung development. METHODS: For comparative studies, immunohistochemical investigations of different experimental fibrosis models were performed. RESULTS: For several epithelial proteins, such as the CD44 adhesion molecule, the enzymes carbanhydrase II and cathepsin D, as well as the lectin galectin-3, a transient epithelial immunoreactivity have been detected. What all four examples have in common is that a part of the foetal antigenic profile reappears under conditions of injury and during the development of pulmonary fibrosis. CONCLUSIONS: The re-expression of foetal antigens in fibrotic samples with a spatio-temporal pattern, as detected by immunocytochemical techniques, indicates that some mechanisms or factors exist, which similarly regulate the differentiation of the epithelium during ontogenesis and in the remodelling process during fibrogenesis.
Animals↗