Human Embryonic Pulmonary Fibroblast Research Articles

Arsenic trioxide-eluting electrospun nanofiber-covered self-expandable metallic stent reduces granulation tissue hyperplasia in rabbit trachea

Stent-related granulation tissue hyperplasia is a major complication that limits the application of stents in airways. In this study, an arsenic trioxide-eluting electrospun nanofiber-covered self-expandable metallic stent (ATO-NFCS) was developed. Poly-L-lactide-caprolactone (PLCL) was selected as the drug-carrying polymer. Stents with two different ATO contents (0.4% ATO/PLCL and 1.2% ATO/PLCL) were fabricated. The in vitro release in simulated airway fluid suggested that the total ATO release time was 1 d. The growth of human embryonic pulmonary fibroblasts (CCC-HPF-1), normal human bronchial epithelial cells and airway smooth muscle cells was inhibited by ATO. When embedded in paravertebral muscle, the nanofiber membrane showed good short-term and long-term biological effects. In an animal study, placement of the ATO-NFCS in the trachea through a delivery system under fluoroscopy was feasible. The changes in liver and kidney function 1 and 7 d after ATO-NFCS placement were within the normal range. On pathological examination, the heart, liver, spleen, lungs and kidneys were normal. The effectiveness of the ATO-NFCS in reducing granulation tissue hyperplasia and collagen deposition was demonstrated in the rabbit airway (n = 18) at 4 weeks. The present study preliminarily investigated the efficacy of the ATO-NFCS in reducing granulation tissue formation in the trachea of rabbits. The results suggest that the ATO-NFCS is safe in vivo, easy to place, and effective for the suppression of granulation tissue formation.

Transcription factor YY1 inhibits the expression of THY1 to promote interstitial pulmonary fibrosis by activating the HSF1/miR-214 axis

Interstitial pulmonary fibrosis (IPF) is a progressive disease of diverse etiology manifesting with proliferation of lung fibroblasts and accumulation of extracellular matrix deposition in pulmonary interstitium. Recent studies show aberrant expression of mRNAs and microRNAs (miRNAs) in human embryonic pulmonary fibroblasts (HEPFs). In this study, we investigated effects of the YY1/HSF1/miR-214/THY1 axis on the functions of HEPFs and IPF. Loss- and gain-of-function tests were conducted to identify roles of YY1, HSF1, miR-214, and THY1 in IPF. As determined by RT-qPCR or western blot assay, silencing YY1 down-regulated HSF1 expression and attenuated the expression of pro-proliferative and fibrosis markers in HEPFs. Meanwhile, viability of HEPFs was impeded by YY1 knockdown. The binding relationship between miR-214 and THY1 was verified using dual-luciferase reporter assay. In HEPFs, down-regulation of HSF1 reduced miR-214 expression to repress proliferation and fibrogenic transformation of HEPFs, while inhibition of miR-214 expression could restrain the fibrogenic transformation property of HEPFs by up-regulating THY1. Subsequently, IPF model in mice was induced by bleomycin treatment. These animal experiments validated the protective effects of YY1 knockdown against IPF-induced lung pathological manifestations, which could be reversed by THY1 knockdown. Our study demonstrates the important involvement of YY1/HSF1/miR-214/THY1 axis in the development of IPF.

Transcription Factor YY1 Inhibits the Expression of THY1 to Promote Interstitial Pulmonary Fibrosis by Activating the HSF1/miR-214 Axis

Background: Pulmonary fibrosis is an inflammatory disease of pulmonary interstitium caused by many reasons. Recent studies have reported aberrant expression of microRNAs (miRNAs) in human embryonic pulmonary fibroblasts (HEPF), but their underlying mechanism remains to be further explored. In this study, we explored effect of YY1/HSF1/miR-214/THY1 axis on interstitial pulmonary fibrosis (IPF). Methods: IPF related microarray GSE24206 was downloaded from GEO database. Loss and gain of function tests were conducted to identify roles of YY1, HSF1, miR-214 and THY1 in IPF. The related expression of related indexes in tissues or cells was measured by qRT-PCR or western blot assay. Cell survival rate was detected by CCK8. Binding relationship between miR-214 and THY1 was verified using dual-luciferase reporter assay. Subsequently, IPF model in mice was induced by bleomycin. And the pathological changes of the lung tissues were assessed by HE and Masson staining, while the amount of collagen in the lung tissues of mice was measured by hydroxyproline assay. Results: In HEPF, YY1 promoted HSF1 and miR-214 expression, while miR-214 could promote the transformation HEPF by downregulating THY1. Silencing YY1 or HSF1 could inhibit the proliferation of HEPF and the expression of Ki67 and biomarkers of fibrosis, while overexpression of HSF1 or miR-214 could reverse this effect. Conclusion: Our study demonstrated the facilitating role of YY1/HSF1/miR-214/THY1 axis in the transformation of IPF. Funding Statement: The authors stated: None. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Animals were treated humanely using approved procedures in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China.

Prodigiosin Alleviates Pulmonary Fibrosis Through Inhibiting miRNA-410 and TGF-β1/ADAMTS-1 Signaling Pathway

Background/Aims: Pulmonary fibrosis is a common outcome of various interstitial lung diseases. Prodigiosin (PG) is a series of red pigment with methoxypyrrole ring. This studyinvestigates therole of prodigiosin in pulmonary fibrosis and its underlying mechanisms. Methods: A pulmonary fibrosis rat model was established by intra-trachealinjection ofbleomycin A5. Rats were divided into 4 groups: Normal group, pulmonary fibrosis Model group, Prodigiosin treatment group and hydrocortisone treatmentgroup. HE and Masson staining were carried outto evaluate histopathological changes. The content of hydroxyproline in lung tissue was determined by alkaline hydrolysis. The expression of PICP and PIIINP was examined by ELISA. The mRNA expression of miR-410, TGF-β1 and ADAMTS1 in lung homogenate were detected by RT-PCR. The bronchoalveolar lavage fluid (BALF) and lung tissues of rats were collected and analyzed. Human embryonic pulmonary fibroblast (HEPF) was used for study in vitro. A dual-luciferase reporter assay was conducted to examine the effect of miR-410 on ADAMTS1 expression. Cell transfection was conducted to inhibit miR-410. MTT assay was performed to investigate cell proliferation. The expressions of miR-410, TGF-β1, ADAMTS1and other fibrosis related biomarkers (Col I, Col III, and α-SMA) wereexamined by RT-PCR and Western Blot. Results: HE and Masson staining showed thickened alveolar septum, hyperplasticcapillaries, and large areas of collagen fiber deposition in pulmonary fibrosis model rats. Rats in prodigiosin and hydrocortisone treatment groups had alleviated symptoms. There was high hydroxyproline expression in model rats, whereas the expression of hydroxyproline reduced after prodigiosin or hydrocortisone treatments. RT-PCR results showed high miR-410,high TGF-β1 and low ADAMTS1 in lung tissue of model rats. The expression of PICP and PIIINP werehigher in BALF of model group than in treatment groups. Prodigiosin and hydrocortisone treatment significantly reduced PICP and PIIINP content. RT-PCR and Western Blot analysis showed that prodigiosin inhibited expression of miR-410 and TGF-β1, but up-regulated ADAMTS1 expression. MTT assay indicated that prodigiosin inhibited HEPF proliferation induced by miR-410 overexpression. Conclusion: Prodigiosin down-regulates the expression of miR-410 and TGF-β1, up-regulates ADAMTS1, leading to decrease accumulation of fibrotic proteins. It could be used in alleviating pulmonary fibrosis.

Compensatory effects of hOGG1 for hMTH1 in oxidative DNA damage caused by hydrogen peroxide

ObjectiveTo investigate the potential compensatory effects of hOGG1 and hMTH1 in the repair of oxidative DNA damage. MethodsThe hOGG1 and hMTH1 gene knockdown human embryonic pulmonary fibroblast cell lines were established by lentivirus-mediated RNA interference. The messenger RNA (mRNA) levels of hOGG1 and hM1TH1 were analyzed by the real-time polymerase chain reaction, and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) formation was analyzed in a high-performance liquid chromatography-electrochemical detection system. ResultsThe hOGG1 and hMTH1 knockdown cells were obtained through blasticidin selection. After transfection of hOGG1 and hMTH1 small interfering RNA, the expression levels of the mRNA of hOGG1 and hMTH1 genes were decreased by 97.2% and 96.2%, respectively. The cells then were exposed to 100μmol/L of hydrogen peroxide (H2O2) for 12h to induce oxidative DNA damage. After H2O2 exposure, hMTH1 mRNA levels were increased by 25% in hOGG1 gene knockdown cells, whereas hOGG1 mRNA levels were increased by 52% in hMTH1 gene knockdown cells. Following the treatment with H2O2, the 8-oxo-dG levels in the DNA of hOGG1 gene knockdown cells were 3.1-fold higher than those in untreated HFL cells, and 1.67-fold higher than those in H2O2-treated wild-type cells. The 8-oxo-dG levels in hMTH1 gene knockdown cells were 2.3-fold higher than those in untreated human embryonic pulmonary fibroblast cells, but did not differ significantly from those in H2O2-treated wild-type cells. ConclusionOur data suggested that hOGG1 could compensate for hMTH1 during oxidative DNA damage caused by H2O2, whereas hMTH1 could not compensate sufficiently for hOGG1 during the process.

Effect of the Wnt1/β-catenin signalling pathway on human embryonic pulmonary fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease associated with a high rate of mortality, characterised by an accumulation of fibroblasts/myofibroblasts in the fibroblastic foci (FF) and by an excessive deposition of extracellular matrix (ECM) in the lung parenchyma. The pathogenesis of this fatal disorder remains unclear. Previous evidence suggests that myofibroblasts are key effectors of the deposition of ECM. In the present study, human embryonic pulmonary fibroblast (HEPF) cells were incubated with different concentrations of Wnt1. The present study revealed that cell proliferation improved following stimulation using different concentrations of Wnt1 in a concentration-dependent manner. When the concentration exceeded 20µg/l, cell proliferation was significant (P<0.05) and the cell expression of α-SMA, vimentin and collagen I mRNA, as well as protein expression, significantly increased (P<0.05). Bronchoalveolar lavage fluid (BALF) was then obtained from bleomycin (BLM)-induced models of pulmonary fibrosis. HEPF cells were cultured with Dulbecco's modified Eagle's medium plus BALF. The mRNA and protein expression of α-SMA, vimentin and collagen I significantly increased and these increases were associated with β-catenin. Furthermore, following being infected with the lentivirus expressing β-catenin shRNA, HEPF cells were cultured with BALF. However, the mRNA and protein expression of α-SMA, vimentin and collagen I did not increase significantly. The present study suggested that the Wnt1/β-catenin signalling pathway can promote HEPF cell proliferation and induced HEPF cells can change into myofibroblasts and promote ECM deposition. These findings may provide a theoretical basis for the treatment of IPF.

Reversal of il-1β-mediated human embryonic pulmonary fibroblast transdifferentiation by targeting the ERK signaling pathway

The aim of the present study was to determine whether Interleukin (IL)-1?-mediated human embryonic pulmonary fibroblast transdifferentiation could be reversed by targeting of the ERK signaling pathway. The human embryonic pulmonary fibroblast MRC-5 cell line was used as a model to observe IL-1?-mediated transdifferentiation as well and the inhibitory effects of lentinan (LNT). Cell proliferation was examined by a CCK-8 assay. ERK signaling activity was detected using immunoblotting with phospho-ERK antibody. The expression levels of fibronectin (FN), Col I and ?-smooth muscle actin (?-SMA) were assessed by either reverse transcription PCR or the SABC assay. IL-1?-induced-ERK signaling activation in MRC-5 cells was inhibited by pretreatment with the LNT or ERK inhibitor U0126. IL-1?-enhanced cell proliferation and expression of FN, Col I and ?-SMA were also attenuated by the treatment with LNT. Our study revealed that activation of ERK signaling is involved in IL-1?-mediated human embryonic pulmonary fibroblast proliferation, phenotypic switching and collagen secretion. These transdifferentiation events in MRC-5 cells could be reversed with LNT treatment by targeting the ERK signaling pathway.

Human mannose‐binding lectin inhibits human cytomegalovirus infection in human embryonic pulmonary fibroblast

A limited number of drugs have been used for treatment of human cytomegalovirus (HCMV), all sharing the similar antiviral mechanism of inhibiting virus replication. This study investigates the anti-HCMV activities of mannose-binding lectin (MBL) from blocking virus entry and inhibiting virus spread. Recombinant human MBL was produced in CHO cells and native human MBL was isolated from human serum. A HCMV neutralization test was performed by pre-treating HCMV with each diluted MBL solution. Then the treated HCMV was inoculated onto the human embryonic pulmonary fibroblasts (HELF), which was followed by HCMV-DNA detection, PP65 positivity examination and confocal imaging of the infected cells. To test the activity of MBL in inhibiting viral spreading after viral invasion, HCMV growth inhibition test was performed. The infected cells were incubated with each diluted MBL, every 24 h, the supernatant was tested for HCMV-DNA. After 72 h, cells were collected for HCMV-DNA and PP65 examination. Then the cytopathic effect was observed and cell viability was measured at the 5 days after infection. HCMV neutralization test revealed 10 μg/mL MBL significantly decreased the HCMV invasion in HELF and the anti-HCMV activity can be blocked by 20 mg/mL mannan. HCMV growth inhibition test indicated that at 48 h after HCMV invasion, the HCMV-DNA level in the culture supernatant with 10 μg/mL MBL was lower than the control. After 72 h, both the HCMV-DNA levels and PP65 positivity in cells incubated with MBL were reduced. This is the first to report on the anti-HCMV activities of MBL by in vitro studies.

Epigenetic enhancement of p66Shc during cellular replicative or premature senescence

The cytoplasmic protein p66Shc is expressed in a wide range of cell types, initially believed to be involved in signaling pathways that regulate cell growth and oxidative stress. Here the epigenetic alterations in the promoter of p66Shc were investigated in replicative senescence and in premature senescence induced by hydrogen peroxide in human embryonic pulmonary fibroblast cells. In both cases p66Shc expression was elevated compared to that seen in growing cultures. However, methylation-specific PCR and bisulfite sequencing revealed that the CpG sites were hypermethylated in all cultures. In addition, quantitative chromatin immunoprecipitation showed increased histone H4 acetylation and histone H3 Lys-4 methylation during replicative senescence, while the increased acetylation of histone H3 and H4, as well as increased H3 Lys-4 methylation, was seen in premature senescence persistence. These findings suggest that histone modifications of p66Shc might be the molecular event in cellular senescence. Taken together, the epigenetic enhancement of p66Shc is associated with the specifically increased histone acetylation and methylation, which may contribute to cellular replicative senescence or premature senescence.

Reversal of Nicotine-Induced Alveolar Lipofibroblast-to-Myofibroblast Transdifferentiation by Stimulants of Parathyroid Hormone-Related Protein Signaling

Nicotine exposure disrupts the parathyroid hormone-related protein (PTHrP)-driven alveolar epithelial-mesenchymal paracrine-signaling pathway, resulting in the transdifferentiation of pulmonary lipofibroblasts (LIFs) to myofibroblasts (MYFs), which seems to be central to altered pulmonary development and function in infants born to mothers who smoke during pregnancy. Modulation of PTHrP-driven signaling can almost completely prevent nicotine-induced LIF-to-MYF transdifferentiation. However, once this process has occurred, whether it can be reversed is not known. Our objective was to determine if nicotine-induced LIF-to-MYF transdifferentiation could be reversed by specifically targeting the PTHrP-mediated alveolar epithelial-mesenchymal paracrine signaling. WI38 cells, a human embryonic pulmonary fibroblast cell line, were initially treated with nicotine for 7 days and LIF-to-MYF transdifferentiation was confirmed by determining the downregulation of the key lipogenic marker, peroxisome proliferator-activated receptor gamma (PPARgamma) and upregulation of the key myogenic marker, alpha-smooth muscle actin (alphaSMA). Because downregulation of the PPARgamma signaling pathway is the key determinant of LIF-to-MYF transdifferentiation, cells were treated with three agonists of this pathway, PTHrP, dibutryl cAMP (DBcAMP), or rosiglitazone (RGZ) for 7 days, and the expression of the PTHrP receptor, PPARgamma, alphaSMA, and calponin was determined by Western analysis and immunohistochemistry. Simultaneously, fibroblast function was characterized by measuring their capacity to take up triglycerides. Nicotine-induced LIF-to-MYF transdifferentiation was almost completely reversed by treatment with RGZ, PTHrP, or DBcAMP, as determined by protein and functional assays. Using a specific molecular approach and targeting specific molecular intermediates in the PTHrP signaling pathway, to our knowledge, this for the first time, demonstrates the reversibility of nicotine-induced LIF-to-MYF transdifferentiation, suggesting not only the possibility of prevention but also the potential for reversal of nicotine-induced lung injury.

PREVENTION OF IN UTERO NICOTINE-INDUCED LUNG INJURY BY MODULATING THE WINGLESS/INT AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ SIGNALING PATHWAYS.

Background In utero nicotine exposure disrupts the critical homeostatic pulmonary epithelial-mesenchymal paracrine signaling pathway, resulting in the transdifferentiation of alveolar intersitial fibroblasts (AIFs) to myofibroblasts (MYFs). AIF-to-MYF transdifferentiation is characterized by down-regulation of the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling pathway and up-regulation of the Wingless/Int (Wnt) signaling pathway. Although effects of in utero nicotine exposure on PPAR-γ signaling are known, there is no information regarding its effect on Wnt signaling. We hypothesize that in utero nicotine exposure up-regulates AIF Wnt signaling, and in utero nicotine-induced lung injury can be prevented by specific molecular targeting to down-regulate Wnt signaling and/or up-regulate PPAR-γ signaling. Objectives To determine (1) how in utero nicotine exposure affects AIF Wnt signaling and (2) if Wnt signaling antagonists and/or PPAR-γ agonists can prevent in utero nicotine-induced AIF-to-MYF transdifferentiation. Methods WI38 cells, a human embryonic pulmonary fibroblast cell line, were cultured using standard methods. At near-confluence, the cells were treated twith nicotine (10−9 M) with or without a Wnt pathway antagonist (Wnt inhibitory factor [WIF-1]), or a PPAR-γ agonist (rosiglitazone [RGZ] 10−5M) for 24 hours to 7 days. Then the expression of various intermediates in Wnt and PPAR-γ signaling was determined at the mRNA (RT-PCR) and protein (Western blot) levels. Results Nicotine treatment up-regulated Wnt pathway signaling intermediates such as LEF-1, β-catenin, its downstream target α-smooth muscle actin (α-SMA), and down-regulated PPAR-γ pathway signaling intermediates such as PPAR-γ and adipocyte differentiation-related protein. Concomitant treatment with WIF-1 or RGZ completely inhibited the nicotine-induced up-regulation of the expression of LEF-1 and its downstream target α-SMA. Conclusions By either down-regulating AIF Wnt signaling and/or by up- regulating AIF PPAR-γ signaling, prohomeostatic AIFs can be maintained, preventing MYF transdifferentiation, thereby potentially preventing in utero nicotine-induced lung injury. Supported by grants from TRDRP ((14RT-0073 and 15IT-0250), Philip Morris USA Inc. and Philip Morris International, and NIH (HL55268 and HL075405).

Mechanism of nicotine-induced pulmonary fibroblast transdifferentiation

We tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary interstitial lipofibroblast (LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in altered pulmonary development and function. Studies were done to determine whether nicotine induces LIF-to-MYF transdifferentiation and to elucidate underlying molecular mechanism(s) involved and to determine whether nicotine-induced LIF-to-MYF transdifferentiation could be prevented by stimulating specific alveolar interstitial fibroblast lipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potent PPARgamma stimulant rosiglitazone (RGZ) for 7 days. Expression of key lipogenic and myogenic markers was examined by RT-PCR, Western hybridization, and immunohistochemistry. The effect of nicotine on triglyceride uptake by WI38 cells and PTHrP binding to its receptor was also determined. Finally, the effect of transfecting WI38 cells with a PPARgamma expression vector on nicotine-induced LIF-to-MYF transdifferentiation was determined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating nicotine-induced LIF-to-MYF transdifferentiation. This was accompanied by decreased PTHrP receptor binding to its receptor. The nicotine-induced LIF-to-MYF transdifferentiation was completely prevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressing PPARgamma. Our data suggest nicotine induces alveolar LIF-to-MYF transdifferentiation through a mechanism involving downregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, which can be prevented using specific molecular targets. Potential therapeutic implications of these observations against in utero nicotine-induced lung injury remain to be tested.

Cloning of human telomerase catalytic subunit gene promoter and studying on its specific transcriptional activities in human lung cancer cells

To clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell. hTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection. Electrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line. The hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.

Effect of melanins from black yeast fungi on proliferation and differentiation of cultivated human keratinocytes and fibroblasts.

The effects of melanin preparations from black yeast fungi (BYF) on the proliferation and differentiation of normal cultivated human skin keratinocytes and embryonic pulmonary fibroblasts have been investigated. Melanin preparations in the range of 5-0.1 microg/ml were optimally active, with a more pronounced effect on keratinocyte than on fibroblast proliferation. Of 17 dihydroxynaphthalene (DHN) natural melanin preparations and two commercial dihydroxyphenylalanine (DOPA) melanin preparations, only one preparation--DOPA melanin (of animal origin) significantly stimulated proliferation of keratinocytes at 5 microg/ml; four preparations (DHN melanin from BYF) significantly inhibited proliferation of these cells at 5 or 1 microg/ml. The remaining preparations had no significant effect. Similarly, of the 17 preparations of DHN melanin from BYF, one preparation significantly stimulated fibroblast proliferation, and four significantly inhibited proliferation at 5 microg/ml, one at all the concentrations, and three from 1 down to 0.1 microg/ml. These melanin preparations were also shown to affect the in vitro differentiation of keratinocytes.