Effects of transdifferentiation and EGF on claudin isoform expression in alveolar epithelial cells.

Rat alveolar epithelial type II cells grown on polycarbonate filters form high-resistance monolayers and concurrently acquire many phenotypic properties of type I cells. Treatment with EGF has previously been shown to increase transepithelial resistance across alveolar epithelial cell (AEC) monolayers. We investigated changes in claudin expression in primary cultured AEC during transdifferentiation to the type I cell-like phenotype (days 0, 1, and 8), and on day 5 in culture +/- EGF (10 ng/ml) from day 0 or day 4. Claudins 4 and 7 were increased, whereas claudins 3 and 5 were decreased, on later compared with earlier days in culture. Exposure to EGF led to increases in claudins 4 and 7 and decreases in claudins 3 and 5. Claudin 1 was only faintly detectable in freshly isolated type II cells and remained unchanged over time in culture and after exposure to EGF. These results suggest that increases in transepithelial resistance accompanying AEC transdifferentiation and/or EGF exposure are mediated, at least in part, by changes in the pattern of expression of specific claudin isoforms.     

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

Aims

The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.

Main methods

Real-time PCR analysis, uptake study of [³H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.

Key findings

The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [³H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [³H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [³H]Gly-Sar uptake were observed in type I-like cells. The maximal [³H]Gly-Sar uptake was observed at pH 6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [³H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC₅₀ value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [³H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.

Significance

PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.     

Gene expression during lens transdifferentiation from pigmented epithelial cells

Pigmented epithelial cells of chicken and human dedifferentiate in the medium containing phenylthiourea and testicular hyaluronidase, and then trans-differentiate into lens cells in vitro. To understand the molecular mechanisms of transdifferentiation, gene expression during lens transdifferentiation was analyzed. As the first step, pigment cell and lens specific genes were isolated and expression of these gene was analyzed by Northern blotting . These results clearly shown that lens transdifferentiation proceeds via neutral cell state in which both pigment and lens specific genes are repressed. Oncogene expression was also analyzed. An elevated expression of the c-myc gene was observed during dedifferentiation process. It is expected that elevated expression of c-myc gene might prevent the cells from entering the G0 phase and thus lead to dedifferentiated state.     

Effects of hypochlorite on cultured respiratory epithelial cells

Neutrophils and eosinophils are involved in the pathogenesis of many respiratory diseases. The enzymes myeloperoxidase and eosinophil peroxidase catalyze the reaction of H₂O₂ with Cl to produce the reactive oxygen species HOCl.

Normal human bronchial epithelial (NHBE) cells were exposed to 0.18-0.90 mM HOCl for 48 h, and studied with immunohistochemical, metabolic and morphological studies.

The ability of the cells to attach to each other and/or to the matrix was altered. Immunohistochemical studies showed a decreased amount of desmosomes and focal adhesion sites, although the morphology of the cells was not affected. The ability of the mitochondria to oxidize glucose was reduced. HOCl-exposed cells had an increased production of NO, probably by an increased activity of cNOS, due to increased intracellular Ca²⁺. The antioxidant N-acetylcysteine inhibited both the NO production and the effects of HOCl on glucose oxidation. The cNOS-inhibitor N-propyl-L-arginine inhibited HOCl-induced NO production. X-ray microanalysis showed an increase in the intracellular Na⁺/K⁺ ratio, which indicates cell damage.

In conclusion, exposure to HOCl results in cell detachment and metabolic alterations in normal human bronchial epithelial cells. Oxygen radicals could in part mediate the effects. Oxygen radicals could hence contribute to the observed epithelial damage in respiratory diseases.     

Effects of hypochlorite on cultured respiratory epithelial cells

Neutrophils and eosinophils are involved in the pathogenesis of many respiratory diseases. The enzymes myeloperoxidase and eosinophil peroxidase catalyze the reaction of H₂O₂ with Cl to produce the reactive oxygen species HOCl.

Normal human bronchial epithelial (NHBE) cells were exposed to 0.18–0.90 mM HOCl for 48 h, and studied with immunohistochemical, metabolic and morphological studies.

The ability of the cells to attach to each other and/or to the matrix was altered. Immunohistochemical studies showed a decreased amount of desmosomes and focal adhesion sites, although the morphology of the cells was not affected. The ability of the mitochondria to oxidize glucose was reduced. HOCl-exposed cells had an increased production of NO, probably by an increased activity of cNOS, due to increased intracellular Ca²⁺. The antioxidant N-acetylcysteine inhibited both the NO production and the effects of HOCl on glucose oxidation. The cNOS-inhibitor N-propyl-L-arginine inhibited HOCl-induced NO production. X-ray microanalysis showed an increase in the intracellular Na⁺/K⁺ ratio, which indicates cell damage.

In conclusion, exposure to HOCl results in cell detachment and metabolic alterations in normal human bronchial epithelial cells. Oxygen radicals could in part mediate the effects. Oxygen radicals could hence contribute to the observed epithelial damage in respiratory diseases.     

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.     

Effects of hyperoxia on DNA synthesis in cultured porcine aortic endothelial cells

The mode of action of hyperoxia on the inhibition of DNA synthesis from thymidine (dThd) was studied in primary cultures of porcine aortic endothelial cells (EC) at confluence. A significant effect of hyperoxia on dThd uptake was detected only after a 48-h exposure to 95% O2. On the other hand, decrease in dThd kinase activity was already observed after a 12-h exposure, and the time course of its reduction followed closely that of the inhibition of dThd incorporation into DNA. The incorporation of dThd triphosphate into DNA in permeabilized EC was unaffected by hyperoxia. Determination of DNA alpha- and beta-polymerase activities showed that hyperoxia reduced the activity of the alpha-polymerase and increased that of the beta-polymerase. We conclude that most of the O2 effects on DNA synthesis from dThd can be attributed to dThd kinase inhibition. The increased activity of DNA beta-polymerase, an enzyme involved in DNA repair, also supports the view that hyperoxia could damage DNA.     

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.     

Effects of hyperoxia on alveolar permeability of neutropenic rabbits

We investigated whether neutrophil suppression would prevent the early hyperoxic injury to the rabbit alveolar epithelium. Rabbits received a single intravenous injection of either nitrogen mustard (2 mg/kg) or saline and were exposed to 100% C2 for 64 h. At the end of the hyperoxic exposure, there were 20 +/- 7 neutrophils/ml blood in the nitrogen mustard group vs. 5,935 +/- 1,174 in the control group (means +/- SE; P less than 0.05). The corresponding numbers in lung extravascular tissue, expressed per high-power field, were 0.37 +/- 7 and 5.9 +/- 0.35, respectively (P less than 0.05). At this time, the rate constants of solute flux for 57Co-vitamin B12 (r = 6.5 A) and 131I-cytochrome c (r = 17 A), across the alveolar epithelium, were 33 +/- 5 (min-1) and 7 +/- 2 for the nitrogen mustard and 29 +/- 5 and 6 +/- 1 for the saline group, respectively. These variables were ninefold higher than their corresponding values in animals breathing air. We concluded that neutrophils do not play a significant role during the early stages of sublethal hyperoxic injury to rabbit alveolar epithelium.     

Effects of hyperosmotic stress on cultured airway epithelial cells

Inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions is unclear. The relation between osmolarity and permeability changes was examined in an airway cell line by the addition of NaCl, NaBr, LiCl, mannitol, or xylitol (295–700 mOsm). Transepithelial resistance was measured as an indicator of the tightness of the cultures. Cell-cell contacts and morphology were investigated by immunofluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. The electrolyte solutions caused a significant decrease in transepithelial resistance from 450 mOsm upwards, when the hyperosmolar exposure was gradually increased from 295 to 700 mOsm; whereas the nonelectrolyte solutions caused a decrease in transepithelial resistance from 700 mOsm upwards. Old cultures reacted in a more rigid way compared to young cultures. Immuno-fluorescence pictures showed weaker staining for the proteins ZO-1, claudin-4, and plakoglobin in treated samples compared to the control. The ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and electrolyte solutions opened a larger proportion of tight junctions than nonelectrolyte solutions. This study shows that hyperosmotic solutions cause the opening of tight junctions, which may increase the permeability of the paracellular pathway and result in increased transepithelial water transport. This study was supported by the Swedish Asthma and Allergy Association and the Swedish Heart Lung Foundation.     

Effects of bacterial endotoxin on protecting copper-deficient rats from hyperoxia

The administration of very low doses of bacterial endotoxin protects rats during exposure to hyperoxia and is associated with the induction of lung antioxidant enzyme activities. Copper-deficient rats have increased susceptibility to O2 toxicity, which may be related to their decreased lung superoxide dismutase activity (SOD) or decreased plasma ceruloplasmin concentrations. To determine whether endotoxin can protect against hyperoxia in this susceptible model, we exposed copper-deficient and control rats to a fractional inspiratory concentration of O2 greater than 0.95 for 96 h after pretreatment with 500 micrograms/kg of bacterial endotoxin or phosphate-buffered saline (PBS). Mortality in the copper-deficient and control rats given PBS and exposed to O2 for 96 h was 100%. Copper-deficient rats died significantly earlier during the exposure than controls. No mortality occurred in either group treated with endotoxin and hyperoxia despite the decreased activity of copper-dependent enzymes in the copper-deficient rats. Copper-deficient rats treated with endotoxin and exposed to hyperoxia did increase lung Cu-Zn-SOD activity, but activity remained below levels found in air-exposed controls. Mn-SOD activity was found to be induced above air-exposed controls in the copper-deficient rats treated with endotoxin and exposed to hyperoxia. Hyperoxic exposure resulted in a marked increase in plasma ceruloplasmin concentrations in the control rats, but no increases in ceruloplasmin occurred in the copper-deficient animals. Endotoxin protects copper-deficient rats from hyperoxia despite their decreased lung Cu-Zn-SOD activity, and decreased plasma ceruloplasmin.     

Cytological characteristics of cultured epithelial cells from thymomas in BUF/Mna rats

Epithelial cells (ECs) from spontaneously developed thymomas in BUF/Mna rats were cultured, characterized and compared with ECs from normal thymuses. The ECs from thymomas had many more keratin filaments and PAS-positive vesicles in the cytoplasm than ECs from normal thymuses. The size and shape of ECs and their nuclei were heterogeneous and about 20% of ECs from thymomas had more than one nucleus. However, the growth rates and saturation densities of ECs from thymomas in monolayer culture were not markedly different from those of normal thymuses. The ECs from thymomas cultured in soft agar did not form any colonies. The distribution of the numbers of chromosomes found in ECs from thymomas was slightly broader than that in normal ECs, but no specific abnormalities nor marker chromosomes were noted. These findings indicate that ECs from thymomas are abnormal, but suggest that they are not malignant in nature.     

Structural and functional aspects of cultured epididymal epithelial cells isolated from pubertal rats

Acidic epididymal glycoprotein (AEG) and androgen-binding protein (ABP) antisera were used to study functional activities of primary cell cultures of the epididymal epithelium of 20--23-day-old rats. Extensive AEG immunoreactivity was associated with almost all epithelial cells of the distal caput, corpus and cauda epididymidis. ABP immunoreactivity was solely confined to some epithelial cells of the caput epididymidis. AEG and ABP immunoreactive cells were identified as principal cells. Morphological studies of enzymically dispersed aggregates of the epididymal epithelial cells showed that stromal cells were satisfactorily removed and that cell aggregates consisted of a predominant population for cells displaying the morphological characteristics of principal cells. Scanning and transmission electron microscopic studies of cultured epididymal epithelial cells in monolayers demonstrated that microvilli and pit-like invaginations of the cell surface were preserved during the first 7--10 days of culture and then gradually disappeared. Other characteristic subcellular structures such as Golgi apparatus and rough endoplasmic reticulum cisterna were preserved. Electrophoretic analysis of [35S]methionine-labelled secretory polypeptides released by epididymal epithelial cells into the culture medium demonstrated a distinct protein band pattern which differed from that observed in the medium of cultured rat Sertoli cells. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually immature rats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell function.     

In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells

It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in approximately 4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair.