Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real-time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 days. Key hepatocyte genes were evaluated, including the differentiation markers albumin, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 (<50% decrease in expression), ABCC4 (>400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. SIGNIFICANCE STATEMENT: This study reports that although human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and cytochrome P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied toward the evaluation of drug properties requiring an extended period of treatment and evaluation.

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Roles of JNK/Nrf2 Pathway on Hemin-Induced Heme Oxygenase-1 Activation in MCF-7 Human Breast Cancer Cells.

Heme oxygenase-1 (HO-1) is highly induced in various human disease states, including cancer, indicating that HO-1 is an emerging target of cancer therapy. In this study, we investigated that the mechanisms of hemin-induced HO-1 expression and its signaling pathways in human breast cancer cell. We used MCF-7 cells, a human breast cancer cell line. Hemin increased HO-1 expression in MCF-7 cells in a dose- and time-dependent manner. Hemin enhanced HO-1 expression through the activation of c-Jun N-terminal kinases (JNK) signaling pathway. Hemin also induced activation of Nrf2, a major transcription factor of HO-1 expression. These responses in MCF-7 cells were completely blocked by pretreatment with brazilin, a HO-1 regulator. These results indicated that brazilin inhibits hemin-induced HO-1 expressions through inactivation of JNK/Nrf2 in MCF-7 cells. Thus, our findings suggest that HO-1 is an important anticancer-target of brazilin in human breast cancer.

A tractable, simplified ex vivo human skin model of wound infection.

The prevalence of infection in chronic wounds is well documented in the literature but not optimally studied due to the drawbacks of current methodologies. Here, we describe a tractable and simplified ex vivo human skin model of infection that addresses the critical drawbacks of high costs and limited translatability. Wounds were generated from excised abdominal skin from cosmetic procedures and cultured, inoculated with Staphylococcus aureus strain UAMS-1, or under aseptic conditions. After three days, the infected wounds exhibited biofilm formation and significantly impaired reepithelialization compared to the control. Additionally, promigratory and proreparative genes were significantly downregulated, while proinflammatory genes were significantly upregulated, demonstrating molecular characterizations of impaired healing as in chronic wounds. This model allows for a simplified and versatile tool for the study of wound infection and subsequent development of novel therapies.

Fluorescent cell tracer dye permits real-time assessment of re-epithelialization in a serum-free ex vivo human skin wound assay.

Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re-epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re-epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome.

A liver-specific long noncoding RNA with a role in cell viability is elevated in human nonalcoholic steatohepatitis.

Hepatocyte apoptosis in nonalcoholic steatohepatitis (NASH) can lead to fibrosis and cirrhosis, which permanently damage the liver. Understanding the regulation of hepatocyte apoptosis is therefore important to identify therapeutic targets that may prevent the progression of NASH to fibrosis. Recently, increasing evidence has shown that long noncoding (lnc) RNAs are involved in various biological processes and that their dysregulation underlies a number of complex human diseases. By performing gene expression profiling of 4,383 lncRNAs in 82 liver samples from individuals with NASH (n = 48), simple steatosis but no NASH (n = 11), and healthy controls (n = 23), we discovered a liver-specific lncRNA (RP11-484N16.1) on chromosome 18 that showed significantly elevated expression in the liver tissue of NASH patients. This lncRNA, which we named lnc18q22.2 based on its chromosomal location, correlated with NASH grade (r = 0.51, P = 8.11 × 10-7 ), lobular inflammation (r = 0.49, P = 2.35 × 10-6 ), and nonalcoholic fatty liver disease activity score (r = 0.48, P = 4.69 × 10-6 ). The association of lnc18q22.2 to liver steatosis and steatohepatitis was replicated in 44 independent liver biopsies (r = 0.47, P = 0.0013). We provided a genetic structure of lnc18q22.2 showing an extended exon 2 in liver. Knockdown of lnc18q22.2 in four different hepatocyte cell lines resulted in severe phenotypes ranging from reduced cell growth to lethality. This observation was consistent with pathway analyses of genes coexpressed with lnc18q22.2 in human liver or affected by lnc18q22.2 knockdown. CONCLUSION: We identified an lncRNA that can play an important regulatory role in liver function and provide new insights into the regulation of hepatocyte viability in NASH. (Hepatology 2017;66:794-808).

New endoplasmic reticulum stress regulator, Gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury.

OBJECTIVE: The accumulation of unfolded protein in the endoplasmic reticulum (ER) initiates an adaptive stress response, termed the unfolded protein response. Previous studies suggested that ER stress might be involved in the formation of neointima after vascular injury. We recently discovered a novel regulator of ER stress, 78-kDa glucose-regulated protein-interacting protein induced by ER stress (Gipie). The objective of this study was to elucidate the role of Gipie using models of vascular disease. APPROACH AND RESULTS: We investigated the functions of Gipie in cultured vascular smooth muscle cells (VSMCs) and in a vascular injury model of a rat carotid artery. The expression of Gipie was predominantly detected in synthetic VSMCs and to a much lesser extent in contractile VSMCs, which was augmented by treatment with thapsigargin. Gipie knockdown increased the phosphorylation levels of c-Jun N-terminal kinase and the number of apoptotic cells under ER stress. Moreover, Gipie knockdown decreased the mature form of collagen I in synthetic VSMCs. The expression of Gipie was rarely detected in the medial VSMCs of the intact carotid artery, whereas it was detected in most of the neointimal cells and some of the medial VSMCs after balloon injury. Depletion of Gipie in the rat carotid artery attenuated the neointimal thickening, which was accompanied by increased cell death in the neointima. Conversely, overexpression of Gipie augmented the neointimal thickening. CONCLUSIONS: Gipie participates in the ER stress response in VSMCs and plays an important role in neointima formation after vascular injury.

Role of fibroblast-derived factors in the pathogenesis of melasma.

BACKGROUND: The hyperactive melanocytes present in melasma skin are confined to the epidermis, but epidermal ablation to treat melasma pigmentation may lead to disease recurrence and aggravation. Melanocyte function is regulated by interactions between melanocytes and neighbouring cells such as keratinocytes and fibroblasts. Because melasma skin usually shows dermal changes after exposure to sunlight, we hypothesized that sun-damaged fibroblasts might play a crucial role in the pathogenesis of melasma. AIM: In this study, the melanogenic role of primary cultured fibroblasts from human melasma skin was investigated. METHODS: We explored whether primary cultured fibroblasts from melasma tissue have a melanogenic function on cultured human epidermal melanocytes and artificial skin. The cytokine profile derived from fibroblasts and their effect on the pigmented epidermal equivalents were investigated. RESULTS: Fibroblasts from the melasma lesion and perilesional skin increased melanogenesis in cultured human epidermal melanocytes and in artificial skin. Fibroblasts from the melasma lesion and perilesional skin secreted more nerve growth factor (NGF)-ß than those in normal buttock skin, and also increased melanogenesis and the expression level of NGF-ß in cultured human epidermal melanocytes and artificial skin. CONCLUSIONS: These results suggest that fibroblasts may play a role in melanogenesis and the pathogenesis of melasma.

TLR4 activation under lipotoxic conditions leads to synergistic macrophage cell death through a TRIF-dependent pathway.

Macrophage dysfunction in obesity and diabetes may predispose to the development of diabetic complications, such as infection and impaired healing after tissue damage. Saturated fatty acids, such as palmitate, are present at elevated concentrations in the plasma of patients with metabolic disease and may contribute to the pathogenesis of diabetes and its sequelae. To examine the effect of lipid excess on macrophage inflammatory function, we determined the influence of palmitate on LPS-mediated responses in peritoneal macrophages. Palmitate and LPS led to a profound synergistic cell death response in both primary and RAW 264.7 macrophages. The cell death had features of apoptosis and necrosis and was not dependent on endoplasmic reticulum stress, ceramide generation, or reactive oxygen species production. Instead, we uncovered a macrophage death pathway that required TLR4 signaling via TRIF but was independent of NF-κB, MAPKs, and IRF3. A significant decrease in macrophage lysosomal content was observed early in the death pathway, with evidence of lysosomal membrane damage occurring later in the death response. Overexpression of the transcription factor TFEB, which induces a lysosomal biogenic program, rescued the lysosomal phenotype and improved viability in palmitate- and LPS-treated cells. Our findings provide new evidence for cross-talk between lipid metabolism and the innate immune response that converges on the lysosome.

Strategies for immortalization of primary hepatocytes.

The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications.

Cross-talk between the Toll-like receptor 4 and Notch1 pathways augments the inflammatory response in the interstitial cells of stenotic human aortic valves.

BACKGROUND AND PURPOSE: Calcific aortic stenosis is a chronic inflammatory disease, and aortic valve interstitial cells (AVIC) play an important role in valvular inflammation. Whereas AVIC from stenotic aortic valves exhibit an augmented response to Toll-like receptor 4 (TLR4) stimulation, the underlying mechanism is unclear. This study tested the hypothesis that an excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the inflammatory response to lipopolysaccharide (LPS) in AVIC of stenotic valves. METHODS AND RESULTS: Human AVIC were isolated from normal and stenotic leaflets. Nuclear factor kappa-B (NF-κB) activation and production of interleukin-8, monocyte chemoattactrant protein-1, and intercellular adhesion molecule-1 were analyzed after treatment with LPS. The role of Notch1 in the inflammatory response was determined using inhibitor, siRNA, and specific ligand. Cells from diseased valves produced greater levels of chemokines and intercellular adhesion molecule-1 that are associated with enhanced NF-κB activation. Interestingly, diseased cells exhibited augmented Jagged1 release and Notch1 activation after TLR4 stimulation. Inhibition and silencing of Notch1 each resulted in greater suppression of the TLR4-induced inflammatory response in diseased cells. Conversely, activation of Notch1 with a specific ligand, Jagged1, enhanced the LPS-induced inflammatory response in normal AVIC. Further, Notch1 intracellular domain was coimmunoprecipited with the inhibitor of NF-κB kinase after LPS stimulation, and inhibition of Notch1 abrogated the difference in the level of NF-κB activation between diseased and normal cells. CONCLUSION: Notch1 enhances the inflammatory response to TLR4 stimulation in human AVIC through modulating NF-κB activation. Excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the TLR4 response in AVIC of stenotic valves.

Historical foundations of wound healing and its potential for acceleration: dose-response considerations.

This paper provides a detailed historical assessment of the origin and developmental progress of the concept of wound healing and its attempted acceleration from its start in the beginning of the 20th century to approximately 1960. Emphasis is placed on the development of cell culture in the assessment of wound healing and in attempts to validate experimental findings via clinical research. Of particular interest were the observations that wound healing could be accelerated in the 30-50% range with the dose response displaying biphasic characteristics consistent with the hormesis dose-response model. Such findings set the stage for the hormetic dose-response revolution that is occurring within the biological and biomedical sciences, including wound healing, whereby considerable research now supports the capacity for endogenous and exogenous agents to accelerate the process of wound healing and its functional performance.

A novel three-dimensional model system for keloid study: organotypic multicellular scar model.

We developed a three-dimensional organotypic multicellular spheroid scar model to mimic the microenvironment of human keloid tissues. Keloid tissues were cultured for 7 days. Changes in total cellularity and apoptotic index in the primary keloid spheroid cultures were evaluated histologically and with a TUNEL assay, respectively. The expression profiles of transforming growth factor-ß (TGF-ß), collagen I, collagen III, elastin, fibronectin, matrix metalloproteinase-2, and matrix metalloproteinase-9 were examined with immunohistochemistry. In addition, these expression profiles were investigated after treating primary keloid spheroids with triamcinolone acetonide. Cell viability and morphology of ex vivo cultured keloid spheroids were maintained, and the apoptotic index did not increase for up to 1 week in culture. Keloid spheroids cultivated ex vivo retained the major characteristics of keloids, such as high levels of collagen I and TGF-ß expression for up to 7 days. The biological activity of keloids responding to TGF-ß was also maintained during ex vivo culture. Moreover, ex vivo triamcinolone acetonide treatment of cultivated keloid spheroids significantly reduced collagen I, collagen III, elastin, and fibronectin expression levels, in accordance with clinical observations. The three-dimensional organotypic multicellular spheroid keloid culture will allow investigators to study keloid pathogenesis and test potential keloid therapeutic agents.

Leukocyte adhesion deficiency-I variant syndrome (LAD-Iv, LAD-III): molecular characterization of the defect in an index family.

Leukocyte adhesion deficiencies are rare clinical syndromes of impaired host defense that provide novel insights into regulation of immune and inflammatory responses. Leukocyte adhesion deficiency (LAD)-I variant (LAD-Iv), also called LAD-III, is a unique disorder in which inside-out signaling of ß1, ß2, and ß3 integrins on leukocytes and platelets is disrupted, leading to impaired cellular adhesion, recurrent infections, and bleeding. We originally reported the second patient with this disorder to be identified and characterized the adhesive deficiencies and functional phenotype of this subject's leukocytes. Here, we show that the molecular defect in this index subject is a new mutation in FERMT3 (KINDLIN-3) which encodes KINDLIN-3, a cytoskeletal protein that interacts with the cytoplasmic tails of ß1, ß2, and ß3 integrins and is required for inside-out and outside-in signaling of these heterodimers. We also report clinical features and previously unrecognized defects in cells from a new patient, a sibling of the original subject that we described who carries the same FERMT3 mutation. Mutations in FERMT3 have now been shown to be the basis for LAD-Iv/LAD-III in each of the four original patients or families that established this syndrome, including the family that we describe.

Thyroid cancer development and progression: emerging role of cancer stem cells.

Thyroid cancer is the most common endocrine malignancy. Although the majority of thyroid cancers are well differentiated and have a favorable prognosis, a minor proportion are poorly differentiated malignancies, which show an aggressive behavior and are refractory to conventional cancer treatments. The molecular mechanisms underlying thyroid development and progression are incompletely understood. Most of thyroid tumorigenesis models propose that thyroid cancer originates from the normal thyrocytes that, via the accumulation of genetic alterations, acquire a malignant phenotype and the ability to metastatize. However, recent progress in clarifying the molecular mechanisms of thyroid embryogenesis/development and the discovery of fetal/stem-like cells within the thyroid gland, have raised the possibility that thyroid cancer originates from progenitor/stem cells. These cells have the ability to self-renew and to undergo multilineage differentiation, and are resistant to common anticancer treatments. Thyroid progenitor/stem cells have been isolated from thyroid cancer and the normal counterpart. Further insights in the biology of these cells will open new perspectives in terms of prevention, diagnosis and therapy of thyroid cancers, especially those with an aggressive behaviour. More effective protocols for the identification and isolation of thyroid cancer stem cells will allow us to specifically and safely target these cells with the aim to definitely eradicate aggressive thyroid cancers.

Applying Probe Electrospray Ionization Mass Spectrometry to Cytological Diagnosis: A Preliminary Study by Using Cultured Lung Cancer Cells.

OBJECTIVES: Cytology and histology are 2 indispensable diagnostic tools for cancer diagnosis, which are rapidly increasing in importance with aging populations. We applied mass spectrometry (MS) as a rapid approach for swiftly acquiring nonmorphological information of interested cells. Conventional MS, which primarily rely on promoting ionization by pre-applying a matrix to cells, has the drawback of time-consuming both on data acquisition and analysis. As an emerging method, probe electrospray ionization-MS (PESI-MS) with a dedicated probe is capable to pierce sample and measure specimen in small amounts, either liquid or solid, without the requirement for sample pretreatment. Furthermore, PESI-MS is timesaving compared to the conventional MS. Herein, we investigated the capability of PESI-MS to characterize the cell types derived from the respiratory tract of human tissues. STUDY DESIGN: PESI-MS analyses with DPiMS-2020 were performed on various type of cultured cells including 5 lung squamous cell carcinomas, 5 lung adenocarcinomas, 5 small-cell carcinomas, 4 malignant mesotheliomas, and 2 normal controls. RESULTS: Several characteristic peaks were detected at around m/z 200 and 800 that were common in all samples. As expected, partial least squares-discriminant analysis of PESI-MS data distinguished the cancer cell types from normal control cells. Moreover, distinct clusters divided squamous cell carcinoma from adenocarcinoma. CONCLUSION: PESI-MS presented a promising potential as a novel diagnostic modality for swiftly acquiring specific cytological information.

Endothelial progenitor cells and endothelial vesicles - what is the significance for patients with chronic kidney disease?

Endothelial progenitor cells are cells derived from the bone marrow that circulate in the bloodstream and can exhibit phenotypic characteristics of endothelial cells. They are thought to be involved in postnatal vasculogenesis and to potentially help repair injured endothelium. Circulating endothelial cells are mature endothelial cells in the circulation, and endothelial vesicles or microparticles are thought to be derived from the membranes of endothelial cells as a result of injury or activation. Recent research has focused on using these markers of endothelial injury and repair to assess the state of endothelial health. These efforts have been hampered by lack of uniformity in methodology and terminology. Recent developments in flow cytometry techniques have allowed better characterization and definition of these cells. We review the common techniques used to identify and isolate these cells, clinical studies in patients with chronic kidney disease (CKD) where they serve as markers of endothelial health and predictors of outcome, and possible mechanisms of progenitor cell dysfunction in CKD.

Neuroglobin attenuates beta-amyloid neurotoxicity in vitro and transgenic Alzheimer phenotype in vivo.

Neuroglobin (Ngb), a vertebrate globin expressed primarily in neurons, is induced by and protects against neuronal hypoxia and cerebral ischemia. To investigate the spectrum and mechanism of Ngb's neuroprotective action, we studied the effect of transgenic overexpression of Ngb on NMDA and beta-amyloid (Abeta) toxicity in murine cortical neuron cultures in vitro and on the phenotype of Alzheimer's disease (AD) transgenic (APP(Sw,Ind)) mice. Compared with cortical neuron cultures from wild-type mice, cultures from Ngb-overexpressing transgenic (Ngb-Tg mice) were resistant to the toxic effects of NMDA and Abeta(25-35), as measured by polarization of cell membrane lipid rafts, mitochondrial aggregation, lactate dehydrogenase release, and nuclear fragmentation. In addition, compared with APP(Sw,Ind) mice, double-transgenic (Ngb-Tg x APP(Sw,Ind)) mice showed reductions in thioflavin-S-stained extracellular Abeta deposits, decreased levels of Abeta(1-40) and Abeta(1-42), and improved behavioral performance in a Y-maze test of spontaneous alternations. These findings suggest that the spectrum of Ngb's neuroprotective action extends beyond hypoxic-ischemic insults. Ngb may protect neurons from NMDA and Abeta toxicity by inhibiting the formation of a death-signaling membrane complex, and interventions that increase Ngb expression could have therapeutic application in AD and other neurodegenerative disorders.

Role of A disintegrin and metalloprotease-12 in neutrophil recruitment induced by airway epithelium.

Among proteases, metalloproteases are implicated in tissue remodeling, as shown in numerous diseases including allergy. ADAMs (A Disintegrin And Metalloprotease) metalloproteases are implicated in physiologic processes such as cytokine and growth factor shedding, cell migration, adhesion, or repulsion. Our aim was to measure ADAM-12 expression in airway epithelium and to define its role during the allergic response. To raise this question, we analyzed the ADAM-12 expression ex vivo after allergen exposure in patients with allergic rhinitis and in vitro in cultured primary human airway epithelial cells (AEC). Clones of BEAS-2B cells transfected with the full-length form of ADAM-12 were generated to study the consequences of ADAM-12 up-regulation on AEC function. After allergen challenge, a strong increase of ADAM-12 expression was observed in airway epithelium from patients with allergic rhinitis but not from control subjects. In contrast with the other HB-epidermal growth factor sheddases, ADAM-10 and -17, TNF-alpha in vitro increased the expression of ADAM-12 by AEC, an effect amplified by IL-4 and IL-13. Up-regulation of ADAM-12 in AEC increased the expression of alpha3 and alpha4 integrins and to the modulation of cell migration on fibronectin but not on collagen. Moreover, overexpression of ADAM-12 in BEAS-2B enhanced the secretion of CXCL1 and CXCL8 and their capacity to recruit neutrophils. CD47 was strongly decreased by ADAM-12 overexpression, a process associated with a reduced adhesion of neutrophils. These effects were mainly dependent on epidermal growth factor receptor activation. In summary, ADAM-12 is produced during allergic reaction by AEC and might increase neutrophil recruitment within airway mucosa.

Early growth response-1 suppresses epidermal growth factor receptor-mediated airway hyperresponsiveness and lung remodeling in mice.

Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.