Barbara Driscoll

Human Amniotic Fluid Stem Cells Can Integrate and Differentiate into Epithelial Lung Lineages

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein‐injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10‐kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung.

ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2

The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L714-L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-d-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1β-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-d-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 μM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.

Stem/Progenitor Cells in Lung Development, Injury Repair, and Regeneration

At least two populations of epithelial stem/progenitor cells give rise to the lung anlage, comprising the laryngo-tracheal complex versus the distal lung below the first bronchial bifurcation. Amplification of the distal population requires FGF9-FGF10-FGFR2b-Sprouty signaling. Residual pools of adult stem cells are hypothesized to be the source of lung regeneration and repair. These pools have been located within the basal layer of the upper airways, within or near pulmonary neuroendocrine cell rests, at the bronchoalveolar junction as well as within the alveolar epithelial surface. Rapid repair of the denuded alveolar surface after injury is clearly key to survival. Strategies to enhance endogenous alveolar epithelial repair could include protection of epithelial progenitors from injury and/or stimulation of endogenous progenitor cell function. Protection with inosine or FGF signaling are possible small molecule therapeutic options. Alternatively, exogenous stem/progenitor cells can be delivered into the lung either intravenously, intratracheally, or by direct injection. Sources of exogenous stem/progenitor cells that are currently under evaluation in the context of acute lung injury repair include embryonic stem cells, bone marrow- or fat-derived mesenchymal stem cells, circulating endothelial progenitors, and, recently, amniotic fluid stem/progenitor cells. Further work will be needed to translate stem/progenitor cell therapy for the lung.

Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

Overexpression of Fibroblast Growth Factor-10 during Both Inflammatory and Fibrotic Phases Attenuates Bleomycin-induced Pulmonary Fibrosis in Mice

RATIONALE Fibroblast growth factor-10 (FGF10) controls survival, proliferation, and differentiation of distal-alveolar epithelial progenitor cells during lung development. OBJECTIVES To test for the protective and regenerative effect of Fgf10 overexpression in a bleomycin-induced mouse model of pulmonary inflammation and fibrosis. METHODS In SP-C-rtTA; tet(O)Fgf10 double-transgenic mice, lung fibrosis was induced in 2-month-old transgenic mice by subcutaneous delivery of bleomycin (BLM), using an osmotic minipump for 1 week. Exogenous Fgf10 expression in the alveolar epithelium was induced for 7 days with doxycycline during the first, second, and third weeks after bleomycin pump implantation, and lungs were examined at 28 days. MEASUREMENTS AND MAIN RESULTS Fgf10 overexpression during Week 1 (inflammatory phase) resulted in increased survival and attenuated lung fibrosis score and collagen deposition. In these Fgf10-overexpressing mice, an increase in regulatory T cells and a reduction in both transforming growth factor-beta(1) and matrix metalloproteinase-2 activity were observed in bronchoalveolar lavage fluids whereas the number of surfactant protein C (SP-C)-positive, alveolar epithelial type II cells (AEC2) was markedly elevated. Analysis of SP-C and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end labeling) double-positive cells and isolation of AEC2 from lungs overexpressing Fgf10 demonstrated increased AEC2 survival. Expression of Fgf10 during Weeks 2 and 3 (fibrotic phase) showed significant attenuation of the lung fibrosis score and collagen deposition. CONCLUSIONS In the bleomycin model of lung inflammation and fibrosis, Fgf10 overexpression during both the inflammatory and fibrotic phases results in a greatly reduced extent of lung fibrosis, suggesting that FGF10 may be useful as a novel approach to the treatment of pulmonary fibrosis.

Lung alveolar integrity is compromised by telomere shortening in telomerase-null mice

Shortened telomeres are a normal consequence of cell division. However, telomere shortening past a critical point results in cellular senescence and death. To determine the effect of telomere shortening on lung, four generations of B6.Cg-Terc(tm1Rdp) mice, null for the terc component of telomerase, the holoenzyme that maintains telomeres, were bred and analyzed. Generational inbreeding of terc-/- mice caused sequential shortening of telomeres. Lung histology from the generation with the shortest telomeres (terc-/- F4) showed alveolar wall thinning and increased alveolar size. Morphometric analysis confirmed a significant increase in mean linear intercept (MLI). terc-/- F4 lung showed normal elastin deposition but had significantly decreased collagen content. Both airway and alveolar epithelial type 1 cells (AEC1) appeared normal by immunohistochemistry, and the percentage of alveolar epithelial type 2 cells (AEC2) per total cell number was similar to wild type. However, because of a decrease in distal lung cellularity, the absolute number of AEC2 in terc-/- F4 lung was significantly reduced. In contrast to wild type, terc-/- F4 distal lung epithelium from normoxia-maintained mice exhibited DNA damage by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) and 8-oxoguanine immunohistochemistry. Western blotting of freshly isolated AEC2 lysates for stress signaling kinases confirmed that the stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) stress response pathway is stimulated in telomerase-null AEC2 even under normoxic conditions. Expression of downstream apoptotic/stress markers, including caspase-3, caspase-6, Bax, and HSP-25, was also observed in telomerase-null, but not wild-type, AEC2. TUNEL analysis of freshly isolated normoxic AEC2 showed that DNA strand breaks, essentially absent in wild-type cells, increased with each successive terc-/- generation and correlated strongly with telomere length (R(2) = 0.9631). Thus lung alveolar integrity, particularly in the distal epithelial compartment, depends on proper telomere maintenance.

Lung cancer stem cells: Progress and prospects

Epithelial stem cells are critical for tissue generation during development and for repair following injury. In both gestational and postnatal stages, the highly branched and compartmentalized organization of the lung is maintained by multiple, resident stem/progenitor cell populations that are responsible for the homeostatic maintenance and injury repair of pulmonary epithelium. Though lung epithelial injury in the absence of oncogenic mutation is more commonly expressed as chronic lung disease, lung cancer is the most common form of death worldwide and poses a highly significant risk to human health. Cancer is defined by the cell of origin, responsible for initiating the disease. The Cancer Stem Cell Hypothesis proposes that cancer stem cells, identified by stem-like properties of self-renewal and generation of differentiated progeny, are responsible for propagating growth and spread of the disease. In lung cancer, it is hypothesized that cancer stem cells derive from several possible cell sources. The stem cell-like resistance to injury and proliferative potentials of bronchioalveolar stem cells (BASCs) and alveolar epithelial type II cells (AEC2), as well as cells that express the cancer stem cell marker glycoprotein prominin-1 (CD133) or markers for side populations make them potential reservoirs of lung cancer stem cells. The abnormal activation of pathways that normally regulate embryonic lung development, as well as adult tissue maintenance and injury repair, including the Wnt, Hedgehog (Hh) and Notch pathways, has also been identified in lung tumor cells. It is postulated that therapies for lung cancer that specifically target stem cell signaling pathways utilized by lung cancer stem cells could be beneficial in combating this disease.

Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

The Milieu of Damaged Alveolar Epithelial Type 2 Cells Stimulates Alveolar Wound Repair by Endogenous and Exogenous Progenitors

Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation.

Amniotic fluid stem cells inhibit the progression of bleomycin-induced pulmonary fibrosis via CCL2 modulation in bronchoalveolar lavage.

The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.