Fate of distal lung epithelium cultured in a decellularized lung extracellular matrix.

Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-ßR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-ßR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix metalloprotease-2 (MMP-2) genes after 1 day in culture (p=0.0135 and 0.0128, respectively) and elevated collagen I expression at 7 days (p=0.0016). These data suggest that the original type II-enriched population underwent a transition to increased expression of mesenchymal markers, perhaps as part of a survival or wound-healing program. These results suggest that additional medium components and/or the application of physiologically appropriate stimuli such as ventilation may be required to promote lung-specific epithelial phenotypes.

Breaking the in vitro alveolar type II cell proliferation barrier while retaining ion transport properties.

Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative "ATI-like" cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a "conditionally reprogrammed cell" phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and "ATI-like" mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air-liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases.

Alveolar epithelial differentiation of human induced pluripotent stem cells in a rotating bioreactor.

Traditional stem cell differentiation protocols make use of a variety of cytokines including growth factors (GFs) and inhibitors in an effort to provide appropriate signals for tissue specific differentiation. In this study, iPSC-derived type II pneumocytes (iPSC-ATII) as well as native isolated human type II pneumocytes (hATII) were differentiated toward a type I phenotype using a unique air-liquid interface (ALI) system that relies on a rotating apparatus that mimics in vivo respiratory conditions. A relatively homogenous population of alveolar type II-like cells from iPSC was first generated (iPSC-ATII cells), which had phenotypic properties similar to mature human alveolar type II cells. iPSC-ATII cells were then cultured in a specially designed rotating culture apparatus. The effectiveness of the ALI bioreactor was compared with the effectiveness of small molecule-based differentiation of type II pneumocytes toward type 1 pneumocytes. The dynamics of differentiation were examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), flow cytometry and immunocytochemistry. iPSC-ATII and hATII cells cultured in the ALI bioreactor had higher levels of type I markers, including aquaporin-5(AQ5), caveolin-1, and T1α, at both the RNA and protein levels as compared with the flask-grown iPSC-ATII and hATII that had been treated with small molecules to induce differentiation. In summary, this study demonstrates that a rotating bioreactor culture system that provides an air-liquid interface is a potent inducer of type I epithelial differentiation for both iPS-ATII cells and hATII cells, and provides a method for large-scale production of alveolar epithelium for tissue engineering and drug discovery.

Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix.

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/ß-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.

Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

Purinergic signaling in wound healing and airway remodeling.

Airway epithelia are continuously damaged by airborne pollutants, pathogens and allergens, and they rely on intrinsic mechanisms to restore barrier integrity. Epithelial repair is a multi-step process including cell migration into the wounded area, proliferation, differentiation and matrix deposition. Each step requires the secretion of various molecules, including growth factors, integrins and matrix metalloproteinases. Evidence is emerging that purinergic signaling promotes repair in human airway epithelia. An injury induces ATP release, which binds P2Y(2) receptors (P2Y(2)Rs) to initiate protein kinase C (PKC)-dependent oxidative activation of TNFα-converting enzyme (TACE), which then releases the membrane-bound ligands of the epidermal growth factor receptor (EGFR). The P2Y(2)R- and EGFR-dependent signaling cascades converge to induce mediator release, whereas the latter also induces cytoskeletal rearrangement for cell migration and proliferation. Similar roles for purinergic signaling are reported in pulmonary endothelial cells, smooth muscle cells and fibroblasts. In chronic airway diseases, the aberrant regulation of extracellular purines is implicated in the development of airway remodeling by mucus cell metaplasia and hypersecretion, excess collagen deposition, fibrosis and neovascularization. This chapter describes the crosstalk between these signaling cascades and discusses the impact of deregulated purinergic signaling in chronic lung diseases.

Human alveolar type II cells secrete and absorb liquid in response to local nucleotide signaling.

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na(+) channel-mediated Na(+) absorption and cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion, both significantly regulated by extracellular nucleotides. In addition, we observed in normal AT2 cells an absence of cystic fibrosis transmembrane conductance regulator regulation of epithelial Na(+) channel activity and an absence of expression/activity of reported calcium-activated chloride channels (TMEM16A, Bestrophin-1, ClC2, and SLC26A9), both features strikingly different from normal airway epithelial cells. Measurements of alveolar surface liquid volume revealed that normal AT2 cells: 1) achieved an extracellular nucleotide concentration-dependent steady state alveolar surface liquid height of ∼4 µm in vitro; 2) absorbed liquid when the lumen was flooded; and 3) secreted liquid when treated with UTP or forskolin or subjected to cyclic compressive stresses mimicking tidal breathing. Collectively, our studies suggest that human AT2 cells in vitro have the capacity to absorb or secrete liquid in response to local alveolar conditions.

Nitric oxide and airway epithelial barrier function: regulation of tight junction proteins and epithelial permeability.

Acute airway inflammation is associated with enhanced production of nitric oxide (NO(.)) and altered airway epithelial barrier function, suggesting a role of NO(.) or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o- cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2-/- mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H2O2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity.

Nitric oxide regulation of MMP-9 activation and its relationship to modifications of the cysteine switch.

Matrix metalloproteases (MMPs) are Zn-containing endopeptidases involved in the degradation of extracellular matrix components and are typically secreted in a latent (pro-MMP) form and activated either by proteolytic or oxidative disruption of a conserved cysteine switch. Several recent studies have suggested that nitric oxide (NO) can contribute to the activation of MMPs, but the mechanisms involved are incompletely understood. We investigated the ability of NO to regulate the activation of (pro)MMP-9 using a variety of NO-donor compounds and characterized modifications of the cysteine switch using a synthetic peptide (PRCGVPDLGR) representing the cysteine switch domain of MMP-9. Among the NO-donors used, only S-nitrosocysteine (SNOC) was found to be capable of modest activation of proMMP-9, but S-nitrosoglutathione (GSNO) or the NONOates, DEA-NO, SPER-NO, or DETA-NO, were ineffective. In fact, high concentrations of DETA-NO were found to inhibit MMP-9 activity, presumably by direct interaction with the active-site Zn (2+). Analysis of chemical modifications within the Cys-containing peptide, PRCGVPDLGR, revealed rapid and transient S-nitrosylation by SNOC and GSNO, and formation of mixed disulfides and dimerized peptide as major final products. Similarly, NONOates induced transient S-nitrosylation and primarily peptide dimerization. Coordination of the peptide Cys with a synthetic Zn (2+) complex, to more closely mimic the structure of the active site in proMMP-9, reduced peptide nitrosylation and oxidation by NONOates, but enhanced peptide nitrosylation by SNOC and GSNO. Collectively, our results demonstrate that NO is incapable of directly activating proMMP-9 and that S-nitrosylation of MMP-9 propeptide by NO-donors is unrelated to their ability to regulate MMP-9 activity.

Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha.

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.

Nitric oxide promotes airway epithelial wound repair through enhanced activation of MMP-9.

The airway epithelium provides a protective barrier against inhaled environmental toxins and microorganisms, and epithelial injury initiates a number of processes to restore its barrier integrity, including activation of matrix metalloproteinases such as MMP-9 (92-kD gelatinase B). Airway epithelial cells continuously produce nitric oxide (NO), which has been linked to cell migration and MMP-9 regulation in several cell types, but the importance of epithelial NO in mediating airway epithelial repair or MMP-9 activation is unknown. Using primary or immortalized human bronchial epithelial cells, we demonstrate that low concentrations of NO promote epithelial cell migration and wound repair in an in vitro wound assay, which was associated with increased localized expression and activation of MMP-9. In addition, in HBE1 cells that were stably transfected with inducible NOS (NOS2), to mimic constitutive epithelial NOS2 expression in vivo, NOS inhibition decreased epithelial wound repair and MMP-9 expression. The stimulatory effects of NO on epithelial wound repair and MMP-9 expression were dependent on cGMP-mediated pathways and were inhibited by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase. Inhibition of cGMP-dependent protein kinase (PKG) attenuated NO-mediated epithelial wound closure, but did not affect MMP-9 expression. However, pharmacologic MMP inhibition and siRNA knockdown of MMP-9 expression demonstrated the contribution of MMP-9 to NO-mediated wound closure. Overall, our results demonstrate that NOS2-derived NO contributes to airway epithelial repair by both PKG-dependent and -independent mechanisms, and involves NO-dependent expression and activation of MMP-9.

Airway epithelial cell migration and wound repair by ATP-mediated activation of dual oxidase 1.

The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signal-regulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.

Nitric oxide and reactive nitrogen species in airway epithelial signaling and inflammation.

Nitric oxide (NO(.-)) is produced by many diverse cell types as a cellular or intracellular signaling molecule, by the activation of nitric oxide synthases (NOSs). All three known NOS isoforms are expressed within the respiratory tract and mediate various airway functional properties such as airway smooth muscle tone, ciliary function, epithelial electrolyte transport, and innate host defense. The respiratory epithelium is a major source of NO(.-), in which it regulates normal epithelial cell function and signaling as well as signaling pathways involved in airway inflammation. In addition to its normal physiological properties, increased airway NO(.-) production in inflammatory respiratory tract diseases such as asthma may activate additional signaling mechanisms to regulate inflammatory-immune pathways, and epithelial barrier (dys)function or repair. The biological actions of NO(.-) are controlled at various levels, including mechanisms that regulate NOS localization and activation, and variable oxidative metabolism of NO(.-), resulting in generation of bioactive reactive nitrogen species (RNS). Moreover, in addition to altered production of NO(.-) or RNS, the presence of various target enzymes and/or metabolic regulators of NO(.-)/RNS can be dramatically altered during airway inflammatory conditions, and contribute to alterations in NO(.-)-mediated signaling pathways in disease. This review summarizes current knowledge regarding NO(.-)-mediated epithelial signaling, as well as disease-related changes in airway NOS biology and target enzymes that affect NO(.-)/RNS signaling mechanisms. A detailed understanding of these various changes and their impact on NO(.-) signaling pathways are needed to fully appreciate the contributions of NO(.-)/RNS to airway inflammation and to develop suitable therapeutic approaches based on regulating NO(.-) function.

Comparison of image quality between conventional and low-dose nonenhanced head CT.

BACKGROUND AND PURPOSE: Increasing use of CT for evaluating neurologic disease may expose patients to considerable levels of ionizing radiation. We compared the image quality of low-mAs head CT scans with that of conventional nonenhanced scans. METHODS: Conventional head CT scans were obtained in 20 patients (all >65 years with history of non-CNS malignancy) by using a multidetector technique: 170 mA and 1-second scanning time (ie, 170 mAs), 140 kVp, table speed of 7.5 mm per rotation, pitch of 0.75, section thickness of 5 mm, and field of view of 25 mm. A limited volume helical data acquisition covering four 5-mm-thick images was obtained by using 90 mAs but otherwise the same parameters. Three neuroradiologists visually rated the resulting images for quality in a blinded comparison. Representative 1- to 4-mm(2) regions of interest were chosen in gray matter and white matter locations. Conspicuity and the contrast-to-noise ratio were analyzed. Statistical comparisons were done by using the Student t test. RESULTS: Mean gray matter conspicuity was not significantly different between the 170- and 90-mAs groups (0.39 +/- 0.19 vs 0.41 +/- 0.03, P =.32). Mean gray matter contrast-to-noise ratio was approximately 22% higher with 170 mAs than with 90 mAs (1.77 +/- 0.52 vs 1.39 +/- 0.38, P =.005). All 90-mAs images were rated as having slightly greater image noise than the 170-mAs scans but with sufficient perceived resolution. CONCLUSION: Although 90-mAs head CT images were moderately noisier than 170-mAs images, they were rated as having acceptable diagnostic quality.

Multiple contributing roles for NOS2 in LPS-induced acute airway inflammation in mice.

Acute lung inflammation and injury were induced by intranasal instillation of lipopolysaccharide (LPS) in normal and type 2 nitric oxide synthase (NOS2)-deficient (NOS2-/-) C57BL/6 mice. LPS-induced increases in extravasated airway neutrophils and in lung lavage fluid of TNF-alpha and macrophage inflammatory protein-2 were markedly lower in NOS2-/- than in wild-type mice, indicating that NOS2-derived nitric oxide (NO.) participates in inflammatory cytokine production and neutrophil recruitment. Instillation of LPS also increased total lung lavage protein and induced matrix metalloproteinase-9 and mucin 5AC, as indexes of lung epithelial injury and/or mucus hyperplasia, and increased tyrosine nitration of lung lavage proteins, a marker of oxidative injury. All these responses were less pronounced in NOS2-/- than in wild-type mice. Inhibition of NOS activity also suppressed production of TNF-alpha and macrophage inflammatory protein-2 by LPS-stimulated mouse alveolar MH-S macrophages, and this was restored by NO. donors, illustrating involvement of NO. in macrophage cytokine signaling. Oligonucleotide microarray (GeneChip) analysis of global lung gene expression revealed that LPS inhalation induced a range of transcripts encoding proinflammatory cytokines and chemokines, stress-inducible factors, and other extracellular factors and suppressed mRNAs encoding certain cytoskeletal proteins and signaling proteins, responses that were generally attenuated in NOS2-/- mice. Comparison of both mouse strains revealed altered expression of several cytoskeletal proteins, cell surface proteins, and signaling proteins in NOS2-/- mice, changes that may partly explain the reduced responsiveness to LPS. Collectively, our results suggest that NOS2 participates in the acute inflammatory response to LPS by multiple mechanisms: involvement in proinflammatory cytokine signaling and alteration of the expression of various genes that affect inflammatory-immune responses to LPS.