Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation

Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein. CONCLUSION: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.     

Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis

Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process.     

Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts

Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-β1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis.     

Down-regulation of cytokeratin 14 mRNA in polyoma virus middle T-transformed rat liver epithelial cells.

We have recently shown that rat liver nonparenchymal epithelial cells, such as T51B cells, selectively express cytokeratin (CK) 14 as a partner of CK8 in their intermediate filaments, and we proposed CK14 as a unique cell lineage marker of the liver epithelial cell population (R. Blouin, M-J. Blouin, I. Royal, A. Grenier, A. Loranger, D. R. Roop, and N. Marceau, Differentiation, submitted for publication, 1992). In the present study, T51B-261A (spontaneously transformed) and T51B-261B (aflatoxin B1-treated) clones and clones derived from T51B cells transfected with SV40 large T (LT) and polyoma virus middle T (MT) were used to investigate CK gene expression in nontransformed and transformed liver epithelial cells. T51B-261A, T51B-261B, MT-T51B, and LT/MT-T51B clones all grew in calcium-deficient medium and formed colonies in soft agar, whereas LT-T51B clones did not grow at all in either one of these assays. T51B-261A and T51B-261B clones formed small, slow growing tumors when injected into newborn syngenic rats, whereas the MT-T51B and LT/MT-T51B clones produced rapidly forming, large tumors. There was no effect of cell transformation on CK expression, except in the clones expressing MT, where the CK intermediate filaments were completely lost. Analyses of [35S]methionine incorporation into the Triton-resistant cytoskeleton and of total proteins confirmed that CKs were absent. In contrast, vimentin intermediate filaments remained unaffected in all of the clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clonal expansion of hepatic progenitor cells and differentiation into hepatocyte-like cells

Hepatic progenitor cells (HPCs) in adult liver are promising for treatment of liver diseases. A biliary-derived HPC population in adult mice has been characterized by co-expression of stem cell marker Sry (sex determining region Y)-box 9 (SOX9) and biliary marker cytokeratin 7 (CK7). However, isolation of these HPCs in adult healthy liver without any selection procedures remains a big challenge in this field. Here, by establishing a simple and efficient method to isolate and expand the CK7⁺SOX9⁺ HPCs in vitro as clones, we acquired a stable and largely scalable cell source. The CK7⁺SOX9⁺ progenitor cells were then further induced to differentiate into hepatocyte-like cells with expression of mature hepatocyte markers albumin (Alb) and hepatocyte nuclear factor 4 alpha (HNF4α), both in vitro and in vivo in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor 9 (FGF9). Furthermore, we found that the HPCs are highly responsive to transforming growth factor-beta (TGF-β) signals. Collectively, we identified and harvested a CK7⁺SOX9⁺ progenitor cell population from adult mouse liver by a simple and efficient approach. The exploration of this HPC population offers an alternative strategy of generating hepatocyte-like cells for cell-based therapies of acute and chronic liver disorders.     

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.     

Hepatic myofibroblasts: A heterogeneous population of multifunctional cells in liver fibrogenesis

Hepatic myofibroblasts constitute a heterogenous population of highly proliferative, pro-fibrogenic, pro-inflammatory, pro-angiogenic and contractile cells that sustain liver fibrogenesis and then fibrotic progression of chronic liver diseases of different aetiology to the common advanced-stage of cirrhosis. These α-smooth muscle actin-positive myofibroblast-like cells, according to current literature, mainly originate by a process of activation and trans-differentiation that involves either hepatic stellate cells or fibroblasts of portal areas. Hepatic myofibroblasts can also originate from bone marrow-derived cells, including mesenchymal stem cells or circulating fibrocytes able to engraft chronically injured liver, as well as, in certain conditions, by a process of epithelial to mesenchymal transition involving hepatocytes and cholangiocytes. Hepatic myofibroblasts may have also additional crucial roles in modulating immune response and in the cross talk with hepatic progenitor (stem) cells as well as with malignant cells of either primary hepatocellular carcinomas or of metastatic cancers.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Differential susceptibility to epithelial-mesenchymal transition (EMT) of alveolar, bronchial and intestinal epithelial cells in vitro and the effect of angiotensin II receptor inhibition

The generation of myofibroblasts via epithelial-mesenchymal transition (EMT), a process through which epithelial cells lose their polarity and become motile mesenchymal cells, is a proposed contributory factor in fibrosis of a number of organs. Currently, it remains unclear to what extent epithelia of the upper airways and large intestine are susceptible to this process. Herein, we investigated the ability of model cell lines of alveolar (A549), bronchial (Calu-3) and colonic (Caco-2) epithelial cells to undergo EMT when challenged with transforming growth factor-β1 (TGF-β1) and other pro-inflammatory cytokines. Western blot and immunofluorescence microscopy demonstrated that A549 cells readily underwent EMT, as evidenced by a spindle-like morphology, increase in the mesenchymal marker, vimentin, and down-regulation of E-cadherin, an epithelial marker. In contrast, neither Calu-3 nor Caco-2 cells exhibited morphological changes nor alterations in marker expression associated with EMT. Moreover, whilst stimulation of A549 cells enhanced migration and reduced their proliferative capacity, no such effect was observed in epithelial cell lines of the bronchus or colon. In addition, concomitant treatment of A549 cells with telmisartan, an angiotensin II receptor antagonist with antifibrotic properties, was found to reduce cytokine-induced collagen I production and cell migration, although expression levels of vimentin and E-cadherin remained unaltered. Mechanistically, telmisartan failed to inhibit phosphorylation of Smad2/3. Together, these results, using representative in vitro models of the alveolus, bronchus and colon, tentatively suggest that epithelial cell plasticity and susceptibility to EMT may differ depending on its tissue origin. Furthermore, our investigations point to the beneficial effect of telmisartan in partial abrogation of alveolar EMT.     

Cadherin-11 contributes to pulmonary fibrosis: potential role in TGF-β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.     

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin alpha2beta1(hi) and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 microg/ml insulin (DMEM+10% FBS+Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.     

Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.     

TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

Background

Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT.

Methods

A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA.

Results

The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes.

Conclusion

Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.     

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells

Background

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

Methods

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

Results

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

Conclusions

Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.