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1.  Airway Tissue Plasminogen Activator Prevents Acute Mortality Due to Lethal Sulfur Mustard Inhalation 
Toxicological Sciences  2014;143(1):178-184.
Rationale: Sulfur mustard (SM) is a chemical weapon stockpiled today in volatile regions of the world. SM inhalation causes a life-threatening airway injury characterized by airway obstruction from fibrin casts, which can lead to respiratory failure and death. Mortality in those requiring intubation is more than 80%. No therapy exists to prevent mortality after SM exposure. Our previous work using the less toxic analog of SM, 2-chloroethyl ethyl sulfide, identified tissue plasminogen activator (tPA) an effective rescue therapy for airway cast obstruction (Veress, L. A., Hendry-Hofer, T. B., Loader, J. E., Rioux, J. S., Garlick, R. B., and White, C. W. (2013). Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am. J. Respir. Cell Mol. Biol. 48, 439–447). It is not known if exposure to neat SM vapor, the primary agent used in chemical warfare, will also cause death due to airway casts, and if tPA could be used to improve outcome. Methods: Adult rats were exposed to SM, and when oxygen saturation reached less than 85% (median: 6.5 h), intratracheal tPA or placebo was given under isoflurane anesthesia every 4 h for 48 h. Oxygen saturation, clinical distress, and arterial blood gases were assessed. Microdissection was done to assess airway obstruction by casts. Results: Intratracheal tPA treatment eliminated mortality (0% at 48 h) and greatly improved morbidity after lethal SM inhalation (100% death in controls). tPA normalized SM-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress. Moreover, tPA treatment resulted in greatly diminished airway casts, preventing respiratory failure from airway obstruction. Conclusions: tPA given via airway more than 6 h after exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects, thereby rescuing from respiratory distress and failure. Intra-airway tPA should be considered as a life-saving rescue therapy after a significant SM inhalation exposure incident.
PMCID: PMC4274387  PMID: 25331496
tPA; airway fibrin; sulfur mustard; fibrinolysis; plastic bronchitis
Stem cells (Dayton, Ohio)  2013;31(12):2767-2778.
Tissue specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA)-injury and then returned to normal. The present study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:GFP model to purify basal cells that proliferated infrequently (GFPbright) or frequently (GFPdim) after NA-injury. Both populations contained TSC but TSC were 8.5-fold more abundant in the GFPbright population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally-differentiated basal cells. The ratio of TSC to UBP was 5:1 in the GFPbright population and 1:5 in the GFPdim population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFPbright and GFPdim populations and passaged the clones 7 times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFPdim lineage. Gene expression analysis showed that β-catenin and Notch pathway genes were differentially expressed in freshly-isolated TSC derived from GFPbright and GFPdim populations. We conclude that: 1) TSC and UPB are members of a single lineage; 2) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and 3) an interaction between the β-catenin and Notch pathways regulates the TSC-to-UPB differentiation process.
PMCID: PMC3844014  PMID: 23712882
multipotential stem cells; unipotential basal progenitor; terminal differentiation; Wnt/β-catenin; Notch
3.  Human Tracheobronchial Basal Cells. Normal versus Remodeling/Repairing Phenotypes In Vivo and In Vitro 
Human tracheobronchial epithelial (TBE) basal cells (BCs) function as progenitors in normal tissue. However, mechanistic studies are typically performed in vitro and frequently use BCs recovered from patients who die of nonrespiratory disease. It is not known whether the cadaveric epithelium (1) is undergoing homeostatic remodeling and/or repair, or (2) yields BC clones that represent homeostatic processes identified in tissue. We sought to compare the phenotype of TBE-BCs with that of BCs cultured under optimal clone-forming conditions. TBE pathology was evaluated using quantitative histomorphometry. The cultured BC phenotype was determined by fluorescence-activated cell sorter analysis. Clone organization and cell phenotype were determined by immunostaining. The cadaveric TBE is 20% normal. In these regions, BCs are keratin (K)-5+ and tetraspanin CD151+, and demonstrate a low mitotic index. In contrast, 80% of the cadaveric TBE exhibits homeostatic remodeling/repair processes. In these regions, BCs are K5+/K14+, and a subset expresses tissue factor (TF). Passage 1 TBE cells are BCs that are K5+/TF+, and half coexpress CD151. Optimal clone formation conditions use an irradiated NIH3T3 fibroblast feeder layer (American Type Culture Collection, Frederick, MD) and serum-supplemented Epicult-B medium (Stemcell Technologies, La Jolla, CA). The TF+/CD151− BC subpopulation is the most clonogenic BC subtype, and is enriched with K14+ cells. TF+/CD151− BCs generate clones containing BCs that are K5+/Trp63+, but K14−/CD151−. TF+ cells are limited to the clone edge. In conclusion, clonogenic human TBE BCs (1) exhibit a molecular phenotype that is a composite of the normal and remodeling/reparative BC phenotypes observed in tissue, and (2) generate organoid clones that contain phenotypically distinct BC subpopulations.
PMCID: PMC3931114  PMID: 23927678
basal cell; remodeling; clonogenic frequency; phenotypic plasticity; stem cell
4.  Role of Peroxisome Proliferator-Activated Receptor β/δ and B-Cell Lymphoma-6 in Regulation of Genes Involved in Metastasis and Migration in Pancreatic Cancer Cells 
PPAR Research  2013;2013:121956.
PPARβ/δ is a ligand-activated transcription factor that regulates various cellular functions via induction of target genes directly or in concert with its associated transcriptional repressor, BCL-6. Matrix remodeling proteinases are frequently over-expressed in pancreatic cancer and are involved with metastasis. The present study tested the hypothesis that PPARβ/δ is expressed in human pancreatic cancer cells and that its activation could regulate MMP-9, decreasing cancer cells ability to transverse the basement membrane. In human pancreatic cancer tissue there was significantly higher expression of MMP-9 and PPARβ/δ, and lower levels of BCL-6 mRNA. PPARβ/δ activation reduced the TNFα-induced expression of various genes implicated in metastasis and reduced the invasion through a basement membrane in cell culture models. Through the use of short hairpin RNA inhibitors of PPARβ/δ, BCL-6, and MMP-9, it was evident that PPARβ/δ was responsible for the ligand-dependent effects whereas BCL-6 dissociation upon GW501516 treatment was ultimately responsible for decreasing MMP-9 expression and hence invasion activity. These results suggest that PPARβ/δ plays a role in regulating pancreatic cancer cell invasion through regulation of genes via ligand-dependent release of BCL-6 and that activation of the receptor may provide an alternative therapeutic method for controlling migration and metastasis.
PMCID: PMC3659435  PMID: 23737761
5.  The Idiopathic Pulmonary Fibrosis Honeycomb Cyst Contains A Mucocilary Pseudostratified Epithelium 
PLoS ONE  2013;8(3):e58658.
We previously identified a MUC5B gene promoter-variant that is a risk allele for sporadic and familial Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (IPF/UIP). This allele was strongly associated with increased MUC5B gene expression in lung tissue from unaffected subjects. Despite the strong association of this airway epithelial marker with disease, little is known of mucin expressing structures or of airway involvement in IPF/UIP.
Immunofluorescence was used to subtype mucus cells according to MUC5B and MUC5AC expression and to identify ciliated, basal, and alveolar type II (ATII) cells in tissue sections from control and IPF/UIP subjects. Staining patterns were quantified for distal airways (Control and IPF/UIP) and in honeycomb cysts (HC).
MUC5B-expressing cells (EC) were detected in the majority of control distal airways. MUC5AC-EC were identified in half of these airways and only in airways that contained MUC5B-EC. The frequency of MUC5B+ and MUC5AC+ distal airways was increased in IPF/UIP subjects. MUC5B-EC were the dominant mucus cell type in the HC epithelium. The distal airway epithelium from control and IPF/UIP subjects and HC was populated by basal and ciliated cells. Most honeycombing regions were distinct from ATII hyperplasic regions. ATII cells were undetectable in the overwhelming majority of HC.
The distal airway contains a pseudostratified mucocilary epithelium that is defined by basal epithelial cells and mucus cells that express MUC5B predominantly. These data suggest that the HC is derived from the distal airway.
PMCID: PMC3603941  PMID: 23527003
6.  Roles for β-Catenin and Doxycycline in the Regulation of Respiratory Epithelial Cell Frequency and Function 
The expression of β-catenin–dependent genes can be increased through the Cre recombinase (Cre)–mediated elimination of the exon 3–encoded sequence. This mutant β-catenin is termed DE3, and promotes the expression of β-catenin–dependent genes. Our previous study used the DE3 model to demonstrate that persistent β-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of β-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element–Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the β-catenin reporter transgene TOPGal demonstrated that β-catenin–dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA+/0/TRE-cre+/0/DE3+/+ mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.
PMCID: PMC3262653  PMID: 21852686
Clara cell; doxycycline; Cre recombinase; β-catenin; stereology
7.  A Single Cell Functions as a Tissue-Specific Stem Cell and the In Vitro Niche-Forming Cell 
Tissue-specific stem cell (TSC) behavior is determined by the stem cell niche. However, delineation of the TSC–niche interaction requires purification of both entities. We reasoned that the niche could be defined by the location of the TSC. We demonstrate that a single CD49fbright/Sca1+/ALDH+ basal cell generates rare label-retaining cells and abundant label-diluting cells. Label-retaining and label-diluting cells were located in the rimmed domain of a unique clone type, the rimmed clone. The TSC property of self-renewal was tested by serial passage at clonal density and analysis of clone-forming cell frequency. A single clone could be passaged up to five times and formed only rimmed clones. Thus, rimmed clone formation was a cell-intrinsic property. Differentiation potential was evaluated in air–liquid interface cultures. Homogenous cultures of rimmed clones were highly mitotic but were refractory to standard differentiation signals. However, rimmed clones that were cocultured with unfractionated tracheal cells generated each of the cell types found in the tracheal epithelium. Thus, the default niche is promitotic: Multipotential differentiation requires adaptation of the niche. Because lung TSCs are typically evaluated after injury, the behavior of CD49fbright/Sca1+/ALDH+ cells was tested in normal and naphthalene-treated mice. These cells were mitotically active in the normal and repaired epithelium, their proliferation rate increased in response to injury, and they retained label for 34 days. We conclude that the CD49fbright/Sca1+/ALDH+ tracheal basal cell is a TSC, that it generates its own niche in vitro, and that it participates in tracheal epithelial homeostasis and repair.
PMCID: PMC3175586  PMID: 21131442
8.  Context-Dependent Differentiation of Multipotential Keratin 14–Expressing Tracheal Basal Cells 
Multipotential (MP) differentiation is one characteristic of a tissue-specific stem cell (TSC). Lineage tracing of tracheobronchial basal cells after naphthalene (NA) injury or in the postnatal period demonstrated that basal cells were MP progenitors for Clara-like and ciliated cells. These studies, as well as reports of spatially restricted, label-retaining basal cells, and MP differentiation by human bronchial cells support the hypothesis that a TSC maintained and repaired the tracheobronchial epithelium. However, differences in basal cell phenotype (keratin [K] 5+ versus K14+), age (postnatal versus adult), health status (normal versus injured), and injury type (acid, detergent, NA) limited comparisons among studies and thus diminished the strength of the TSC argument. The finding that K14 was up-regulated after NA injury was a caveat to our previous analysis of reparative (r)K14-expressing cells (EC). Thus, the present study lineage traced steady-state (s)K14EC and evaluated differentiation potential in the normal and repairing epithelium. We showed that sK14EC were unipotential in the normal epithelium and MP after NA, sK14EC-dervied clones were not restricted to putative TSC niches, sK14EC cells were a direct progenitor for Clara-like and ciliated cells, MP-sK14EC clones accumulated over time, and sK14EC-derived Clara-like cells were progenitors for ciliated cells.
PMCID: PMC3175566  PMID: 21131447
basal; clara-like; ciliated; differentiation potential; lineage tracing; tissue-specific stem cell

Results 1-8 (8)