ATI-like Cells Research Articles

Heat-Not-Burn cigarette induces oxidative stress response in primary rat alveolar epithelial cells

There has been an increase in the usage of heat-not-burn (HNB) cigarette products. However, their effects on alveolar epithelial cells (AECs) remain unknown. AECs are the target cells of conventional cigarette smoking-related respiratory diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and lung cancer whose pathogenesis involves oxidative stress. In this study, primary rat AECs were isolated, cultured and stimulated by HNB cigarette smoke extract (CSE). Our data indicate that rat AECs exposed to HNB CSE induced oxidative stress response genes (e.g. Hmox-1, Gsta1, Gsta3 and Nqo1). We also compared the oxidative stress response between two different types of AECs, alveolar type I-like (ATI-like) cells and type II (ATII) cells, and between two different types of cigarette, HNB cigarettes and conventional cigarettes. The expressions of Gsta1, Gsta3 and Nqo1 were higher in ATII cells than ATI-like cells in response to HNB and conventional cigarettes, but there was no significant difference in their expression levels between HNB cigarette and conventional cigarette. Taken together, our results suggest that HNB cigarettes have the similar potential as conventional cigarette products to induce oxidative stress response in AECs.

OCTN2-Mediated Acetyl-l-Carnitine Transport in Human Pulmonary Epithelial Cells In Vitro.

The carnitine transporter OCTN2 is associated with asthma and other inflammatory diseases. The aims of this work were (i) to determine carnitine uptake into freshly isolated human alveolar type I (ATI)-like epithelial cells in primary culture, (ii) to compare the kinetics of carnitine uptake between respiratory epithelial in vitro cell models, and (iii) to establish whether any cell line was a suitable model for studies of carnitine transport at the air-blood barrier. Levels of time-dependent [3H]-acetyl-l-carnitine uptake were similar in ATI-like, NCl-H441, and Calu-3 epithelial cells, whereas uptake into A549 cells was ~5 times higher. Uptake inhibition was more pronounced by OCTN2 modulators, such as l-Carnitine and verapamil, in ATI-like primary epithelial cells compared to NCl-H441 and Calu-3 epithelial cells. Our findings suggest that OCTN2 is involved in the cellular uptake of acetyl-l-carnitine at the alveolar epithelium and that none of the tested cell lines are optimal surrogates for primary cells.

The Role of PGE2 in Alveolar Epithelial and Lung Microvascular Endothelial Crosstalk

Disruption of the blood-air barrier, which is formed by lung microvascular endothelial and alveolar epithelial cells, is a hallmark of acute lung injury. It was shown that alveolar epithelial cells release an unidentified soluble factor that enhances the barrier function of lung microvascular endothelial cells. In this study we reveal that primarily prostaglandin (PG) E2 accounts for this endothelial barrier-promoting activity. Conditioned media from alveolar epithelial cells (primary ATI-like cells) collected from BALB/c mice and A549 cells increased the electrical resistance of pulmonary human microvascular endothelial cells, respectively. This effect was reversed by pretreating alveolar epithelial cells with a cyclooxygenase-2 inhibitor or by blockade of EP4 receptors on endothelial cells, and in A549 cells also by blocking the sphingosine-1-phosphate1 receptor. Cyclooxygenase-2 was constitutively expressed in A549 cells and in primary ATI-like cells, and was upregulated by lipopolysaccharide treatment. This was accompanied by enhanced PGE2 secretion into conditioned media. Therefore, we conclude that epithelium-derived PGE2 is a key regulator of endothelial barrier integrity via EP4 receptors under physiologic and inflammatory conditions. Given that pharmacologic treatment options are still unavailable for diseases with compromised air-blood barrier, like acute lung injury, our data thus support the therapeutic potential of selective EP4 receptor agonists.

Organic cation transporter function in different in vitro models of human lung epithelium

Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air–blood barrier. OCT activity was investigated by measuring [14C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1μM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02μl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.

Prostaglandin Transporter (PGT/SLCO2A1) Protects the Lung from Bleomycin-Induced Fibrosis

Prostaglandin (PG) E2 exhibits an anti-fibrotic effect in the lung in response to inflammatory reactions and is a high-affinity substrate of PG transporter (SLCO2A1). The present study aimed to evaluate the pathophysiological relevance of SLCO2A1 to bleomycin (BLM)-induced pulmonary fibrosis in mice. Immunohistochemical analysis indicated that Slco2a1 protein was expressed in airway and alveolar type I (ATI) and II (ATII) epithelial cells, and electron-microscopic immunohistochemistry further demonstrated cell surface expression of Slco2a1 in ATI cells in wild type (WT) C57BL/6 mice. PGE2 uptake activity was abrogated in ATI-like cells from Slco2a1-deficient (Slco2a1 -/-) mice, which was clearly observed in the cells from WT mice. Furthermore, the PGE2 concentrations in lung tissues were lower in Slco2a1 -/- than in WT mice. The pathological relevance of SLCO2A1 was further studied in mouse BLM-induced pulmonary fibrosis models. BLM (1 mg/kg) or vehicle (phosphate buffered saline) was intratracheally injected into WT and Slco2a1 -/- mice, and BLM-induced fibrosis was evaluated on day 14. BLM induced more severe fibrosis in Slco2a1 -/- than in WT mice, as indicated by thickened interstitial connective tissue and enhanced collagen deposition. PGE2 levels were higher in bronchoalveolar lavage fluid, but lower in lung tissues of Slco2a1 -/- mice. Transcriptional upregulation of TGF-β1 was associated with enhanced gene transcriptions of downstream targets including plasminogen activator inhitor-1. Furthermore, Western blot analysis demonstrated a significant activation of protein kinase C (PKC) δ along with a modest activation of Smad3 in lung from Slco2a1 -/- mice, suggesting a role of PKCδ associated with TGF-β signaling in aggravated fibrosis in BLM-treated Slco2a1 -/- mice. In conclusion, pulmonary PGE2 disposition is largely regulated by SLCO2A1, demonstrating that SLCO2A1 plays a critical role in protecting the lung from BLM-induced fibrosis.

OCTN1‐Mediated Ergothioneine Uptake Protects Lung Epithelial Cells From Tobacco Smoke‐Induced Damage

Smoking is a major contributing factor for chronic obstructive pulmonary disease (COPD). Tobacco smoke exposure causes oxidative stress which results in alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair. Ergothioneine (ET), an amino acid transported by OCTN1, has been shown to exhibit antioxidant and cytoprotective capacities in vitro. Hence, the objective of this study is to understand the role of OCTN1 and the effect of ET on human alveolar cells when subjected to tobacco smoke‐induced oxidative stress.Confocal microscopy and Western blot was used to determine the presence of OCTN1 in A549 cells and freshly isolated human alveolar epithelial type II (ATII) and type I‐like (ATI‐like) cells in primary culture. A549 cells were cultured in medium containing 1 mM of ET and its intracellular accumulation was measured by LC‐MS/MS over a 3‐week period. A549 cells were also was exposed to various concentrations of cigarette smoke extract (CSE). Prior to the experiment, cells were either cultured for 1 week in the presence or absence of 1 mM ET. An MTT assay was used to assess cell viability for the difference exposures to CSE.Immunoblot and CLSM showed OCTN1 expression in A549, ATII and ATI‐like cells. The uptake of ET by A549 epithelial cells was saturated after approximately 7 days of culture. Western blot data supported the CLSM results and also indicated that OCTN1 levels were increased in the presence of ET. The MTT assay showed that cells pre‐treated with ET were less affected by the cytotoxic effects of CSE.These preliminary data indicate that OCTN1 might play a role in the pathogenesis of tobacco smoke‐induced COPD by regulating ergothioneine transport.

Infection of mice with influenza A/WSN/33 (H1N1) virus alters alveolar type II cell phenotype.

Influenza viruses cause acute respiratory disease of great importance to public health. Alveolar type II (ATII) respiratory epithelial cells are central to normal lung function and are a site of influenza A virus replication in the distal lung. However, the consequences of infection for ATII cell function are poorly understood. To determine the impact of influenza infection on ATII cells we used C57BL/6-congenic SP-C(GFP) mice that express green fluorescent protein (GFP) under the control of the surfactant protein-C (SP-C) promoter, which is only active in ATII cells. Most cells isolated from the lungs of uninfected SP-C(GFP) mice were GFP(+) but did not express the alveolar type I (ATI) antigen podoplanin (PODO). ATII cells were also EpCAM(+) and α2,3-linked sialosaccharide(+). Infection with influenza A/WSN/33 virus caused severe hypoxemia and pulmonary edema. This was accompanied by loss of whole lung GFP fluorescence, reduced ATII cell yields, increased ATII cell apoptosis, reduced SP-C gene and protein expression in ATII cell lysates, and increased PODO gene and protein levels. Flow cytometry indicated that infection decreased GFP(+)/PODO(-) cells and increased GFP(-)/PODO(+) and GFP(-)/PODO(-) cells. Very few GFP(+)/PODO(+) cells were detectable. Finally, infection resulted in a significant decline in EpCAM expression by PODO(+) cells, but had limited effects on α2,3-linked sialosaccharides. Our findings indicate that influenza infection results in a progressive differentiation of ATII cells into ATI-like cells, possibly via an SP-C(-)/PODO(-) intermediate, to replace dying or dead ATI cells. However, impaired SP-C synthesis is likely to contribute significantly to reduced lung compliance in infected mice.

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

Insulin-like growth factor-I stimulates differentiation of ATII cells to ATI-like cells through activation of Wnt5a

Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and β-catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using small-interfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway.

Insulin Like Growth Factor‐1α Stimulates Differentiation of ATII Cells to ATI‐Like Cells Through Wnt5a

Alveolar type II (ATII) epithelial cells are important in the repair of the lung following acute lung injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo, and can differentiate into an ATI‐like phenotype in vitro. The mechanisms responsible for transdifferentiation are not well understood. In this study we investigated the mechanisms by which insulin‐like growth factor 1‐α (IGF‐1α), which is increased in bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome, promotes rat ATII to ATI differentiation in primary cultures. Gene expression of IGF‐1α was significantly increased in ATI‐like cells compared with ATII cells. Treatment of ATII cells with recombinant IGF‐1α accelerated the transition of ATII to ATI‐like cells, and this effect was inhibited by the IGF‐1α receptor blocker P40. Knockdown of Wnt5a using siRNA slowed the differentiation, as indicated by changes in the expression of ATII and ATI markers. These results suggest that IGF‐1α promotes differentiation of ATII to ATI cells through the non‐canonical Wnt pathway.

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

BackgroundInfluenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes.MethodsAlveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively.ResultsWe found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels.ConclusionsOur results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.

Human Alveolar Epithelial Cell Injury Induced by Cigarette Smoke

BackgroundCigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases.Methodology/Principal FindingsWe studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE.ConclusionsOur study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.

Type I Alveolar Epithelial Phenotype in Primary Culture

Type I alveolar epithelial cells (ATIs) are very large, thin cells, which extend across several air sacs and cover more than 95% of the alveolar surface area. ATIs are the target of many insults, including ventilator-induced lung injury, and are generally considered terminally differentiated cells arising from type II cell (ATII) lineage. ATIs have proven difficult to harvest and maintain in primary culture, which is why much of ATI biology has been inferred from studies on ex vivo, ATII-derived, so-called ATI-like cells. We report on a modified approach to rat ATI harvest and primary culture, which yielded the following observations: (1) rat ATI can be harvested and maintained with a high degree of purity in primary culture; (2) in vitro growth characteristics of primary ATIs differ from those of ATII-derived ATI-like cells; ATIs, but not ex vivo, ATII-derived ATI-like cells, are capable of cell division; (3) ATIs readily repair plasma membrane wounds without the subsequent loss of their ability to divide; (4) ATI monolayers heal scratch wounds primarily by cell spreading and migration. Although the ability of ATIs to divide may be limited to the in vitro environment, we do believe that their role in alveolar wound repair deserves to be revisited, and the molecular control of ATI-ATII plasticity further explored.

Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells

The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidated lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxides formed by the direct effects of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2, heat shock protein 70, and Fos-related antigen 1 protein expression. Furthermore, we found higher sensitivity of ATI-like cells compared to ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. Understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.

Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung

In order to delineate individual pathomechanisms in acute lung injury and pulmonary toxicology, we developed a primary coculture system to simulate the human alveolo-capillary barrier. Human pulmonary microvascular endothelial cells (HPMEC) were cocultivated with primary isolated human type II alveolar epithelial cells (HATII) on opposite sides of a permeable filter support, thereby constituting a bilayer. Within 7-11 days of coculture, the HATII cells partly transdifferentiated to type-I-like (HATI-like) cells, as demonstrated by morphological changes from a cuboidal to a flattened morphology, the loss of HATII-cell-specific organelles and the increase of HATI-cell-related markers (caveolin-1, aquaporin-5, receptor for advanced glycation end-products). Immunofluorescent analysis detected type-II-like and type-I-like alveolar epithelial cells mimicking the heterocellular composition of alveolar epithelium in vivo. The heterocellular epithelial monolayer showed a circumferential staining of tight-junctional (ZO-1, occludin) and adherens-junctional (E-cadherin, beta-catenin) proteins. HPMEC on the opposite side also developed tight and adherens junctions (VE-cadherin, beta-catenin). Under integral barrier properties, exposure to the proinflammatory cytokine tumour necrosis factor-alpha from either the endothelial (basolateral) or the epithelial (apical) side caused a largely compartmentalized release of the chemokines interleukin-8 and monocyte chemoattractant protein-1. Thus, the established coculture provides a suitable in vitro model to examine barrier function at the distal lung, including the interaction of microvascular endothelial cells with ATII-like and ATI-like epithelial cells. The compartmentalization of the barrier-forming bilayer also allows mechanisms of lung injury to be studied in both the epithelial (intra-alveolar) and the endothelial (intravascular) compartments.

Spectral Monitoring of Surfactant Clearance during Alveolar Epithelial Type II Cell Differentiation

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.

A primary in vitro co‐culture model of the human alveolo‐capillary barrier to study lung injury and repair

Due to an increasing interest in pulmonary toxicology and pathobiology, there is a need to develop cell culture models of the alveolar-capillary barrier. We established a co-culture system of human pulmonary microvascular endothelial cells (HPMEC) and human alveolar epithelial cells, consisting of type II (hATII) and type I-like (hATI-like) cells, on opposite sides of a permeable filter support. Primary isolated hATII cells were seeded on the top surface of collagen typ I- coated Transwell filter units (24-well filter plate) and grown to confluence simultaneously with HPMEC on the lower surface over ten to fourteen days. Within 10 days of co-culture the hATII cells partly transdifferentiated to ATI-like cells, demonstrated through morphological changes from a cuboidal to a flattened morphology of the cells, the loss of cell type specific organells (lamellar bodies) and an increased mRNA expression of RAGE and AQP-5. hATII and hATI-like cells established a contact inhibited monolayer, showing a circumferential staining of the tight junction and the adherens junction proteins (ZO-1, Occludin, E-Cadherin, alpha- and β-Catenin). The generation of a polarized epithelial cell monolayer resulted in trans-monolayer resistance values of 1250±350 Ohm*cm2 and trans-bilayer resistance values of 1730±460 Ohm*cm2 from day 7 to day 10. HPMEC on the opposite side of the permeable 24-well filter plate developed monolayer with tight and adherens junctions. Our primary co-culture model will be suitable to examine interactions of both alveolar epithelium and microvascular endothelium in the pathogenesis of lung injury and the following repair processes.

Temporal dependence of ectopeptidase expression in alveolar epithelial cell culture: implications for study of peptide absorption

There is little data available regarding the extent of peptide metabolism encountered following inhalation to the lung. We have studied the activity of five ectopeptidases in primary rat alveolar epithelial cells, A549 cells and pulmonary macrophages. Peptidase activity was assayed in the plasma membrane fractions (PMF) of primary type II alveolar epithelial cells (ATII cells) after 2 days in culture and after 7 days in culture when they had formed monolayers of type I-like cells (ATI-like cells). Dipeptidyl peptidase IV (DPP) activity fell from 36.65 mU/mg protein to 16.29 mU/mg protein between day 2 and day 7 in culture, aminopeptidase N (AMN) activity increased from 16.16 mU/mg protein to 23.99 mU/mg protein, angiotensin-converting enzyme (ACE) activity was lost (4.29 mU/mg protein at day 2, not detected at day 7), and carboxypeptidase M (CPM) activity was acquired (not detected at day 2, 5.20 mU/mg protein at day 7). The profile of exopeptidase expression in A549 cells was similar to that of primary rat alveolar cells at day 7 in culture (DPP 24.24 mU/mg protein, AMN 47.74 mU/mg protein, CPM 4.28 mU/mg protein, ACE not detected). Macrophages expressed high levels of aminopeptidases (DPP 46.85 mU/mg protein, AMN 28.28 mU/mg protein) but carboxypeptidase activity was not detected. Low neutral endopeptidase 24.11 (NEP) activity was found in all cell types studied (0.96–2.41 mU/mg protein). The qualitative and quantitative changes in the peptidase activity of primary cultured rat alveolar cells between day 2 and day 7 in culture has implications for the use of alveolar cell monolayers as drug absorption models to investigate peptide absorption from the lung. Ectopeptidase activity in cultured alveolar cells can be used to infer the peptide-metabolising capacity of the surface of the alveolar epithelium. The aminopeptidase activity in particular, if representative of enzyme activity in vivo, would offer a significant metabolic barrier to systemic delivery of peptides via the lung.

Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells.

Lung Na-K-ATPase has been shown to contribute to vectorial Na+ transport and edema clearance. The alpha 1- and beta 1-Na-K-ATPase subunits have been localized to alveolar type II (ATII) cells, and the alpha 2-Na-K-ATPase has been reported in rat lung homogenates. Expression of Na-K-ATPase alpha 1-, alpha 2-, and beta 1-subunits was investigated in rat ATII cells cultured for 7 days, a period during which they lose their phenotypic markers and differentiate to an alveolar type I (ATI)-like cell phenotype. Differentiation of ATII cells to an ATI-like phenotype resulted in a decrease of alpha 1- and an increase of alpha 2-mRNA and protein abundance without changes in the beta 1-subunit. Thus ATI-like cells exhibited a mixture of alpha 1- and alpha 2-isoforms. Nuclear run-on analysis suggests that these changes were transcriptionally regulated. The existence of the distinct functional classes of Na-K-ATPase in ATII and ATI-like cells was confirmed by ouabain inhibition of Na-K-ATPase activity. Ouabain inhibition of ATII cells was consistent with expression of the alpha 1-isozyme [50% inhibitory concentration (IC50) = 4 x 10(-5) M], whereas, in ATI-like cells, it was consistent with the presence of both alpha 1- and alpha 2-isozymes (IC50 = 9.0 x 10(-5) and 1.5 x 10(-7) M, respectively); [3H]ouabain binding studies corroborated these findings. Our results indicate that, during ATII cell cytodifferentiation with time in culture, there is a shift in isoform composition that may reflect physiological functions of alveolar epithelial cells.