Proliferation Of Human Lung Fibroblasts Research Articles

Protective role of gambogic acid in experimental pulmonary fibrosis in vitro and in vivo

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive disorder with poor prognosis. The treatment options for IPF are very limited. Gambogic acid (GA) has anticancer effect and anti-proliferative activity which is extracted from a dried yellow resin of the Garcinia hanburyi Hook.f. [Clusiaceae (Guttiferae)] in Southeast Asia. However, the anti-fibrotic activities of GA have not been previously investigated. MethodsIn this study, the effects of GA on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells and endothelial–mesenchymal transition (EndoMT) in human pulmonary microvascular endothelial cells (HPMECs), on the proliferation of human lung fibroblasts (HLF-1) were investigated in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis was investigated in vivo. ResultsIn TGF-β1 stimulated A549 cells, treatment with GA resulted in a reduction of EMT with a decrease in vimentin and p-Smad3 and an increase in E-cadherin instead. In TGF-β1 stimulated HPMECs, treatment with GA resulted in a reduction of EndoMT with a decrease in vimentin, and an increase in VE-cadherin instead. In the hypoxic HPMECs, treatment with GA reduced Vasohibin-2 (VASH-2), whereas increased VASH-1. In TGF-β1 stimulated HLF-1, treatment with GA reduced HLF-1 proliferation with a decrease in platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF-2) expressions. In vivo, treatment with GA for 2 weeks resulted in an amelioration of the BLM-induced pulmonary fibrosis in rats with a lower VASH-2. Instead, it was observed a higher VASH-1 expression at early stage of fibrosis at 1mg/kg, with reductions of the pathological score, collagen deposition, α-SMA, PDGF and FGF-2 expressions at fibrotic stage at 0.5mg/kg and 1mg/kg. ConclusionIn summary, GA reversed EMT and EndoMT, as well as HLF-1 proliferation in vitro and prevented pulmonary fibrosis in vivo by modulating VASH-2/VASH-1 and suppressing the TGF-β1/Smad3 pathway.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells: Importance of ERK1/2 and AKT Signaling Pathways.

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′ -kinase(PI3K)-Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

Abstract 18888: 2-Methoxyestrone Retards the Progression of Angioproliferative Pulmonary Hypertension in Rats - Role of 17beta-Hydroxysteroid Dehydrogenase Pathway

Background: 2-Methoxyestradiol (2ME2; major non-estrogenic metabolite of estradiol) attenuates monocrotaline (MCT)- and SU5416+hypoxia (SU/Hx)-induced pulmonary arterial hypertension (PH). 2ME2 is dehydrogenated by type-2 17β-hydroxysteroid dehydrogenase (17-HSD2) to 2-methoxyestrone (2ME1), a metabolite with no biological activity. However, in vivo 2ME1 may be converted back to 2ME2 by type-1 17β-HSD (17-HSD1). Our recent study indicates that 2ME-2 and all-trans retinoic acid (atRA; a 17HSD1 inducer) have synergistic therapeutic effects in MCT-induced PH.< Methods: To study the role of 17β-HSD metabolic pathway on development of PH, first we examined the effects of 2-ME1 on proliferation of human pulmonary artery smooth muscle cells (hPASMC), lung fibroblasts (hLF) and hela cells, in the presence or absence of atRA. Next, in male rats, we studied the effects of 2ME1 on progression of MCT-induced PH (Day14-28 post MCT). Finally, we investigated the effects of 2ME1 on SU/Hx-induced PH. Ten days into hypoxia, intact (F) and ovariectomized (OVX) female rats were implanted minipumps delivering vehicle (PEG-400 0.5μl/h; F-SU and OVX-SU groups) or 2-ME1 (20μg/kg/h; F-2ME1 and OVX-2ME1 groups) Results: In hPASMC, hLF and hela cells 2ME1 had no/modest antimitogenic effect at a high pharmacological concentration, whereas in the presence of atRA, 2ME1 exhibited strong and concentration-dependent inhibition of cells growth. In male rats, 2ME1 retarded the progression of MCT-PH, and attenuated right ventricle (RV) and pulmonary vascular remodeling. In female rats SU/Hx induced severe angioproliferative PAH (RVPSP: F=23+2 vs F-SU = 116+10mmHg) and 2ME1 reduced PAH. Ovariectomy attenuated PAH (RVPSP: F-SU= 116+10 vs F-2ME1=96+10mmHg and in OVX-SU rats 2ME1 significantly retarded the progression of disease (RVPSP: OVX-SU= 99+9 mmHg vs. OVX-2ME1= 71+4) and markedly reduces RV remodeling (RV/LV+S: OVX-SU=0.715+0.021 vs. OVX-2ME1=0.555+0.026; p<0.001) Conclusions: This study provides the first evidence that in vivo 2ME1 is biologically active and has therapeutic effects in angioproliferative PAH. Presented data suggest that 17β-HSD pathway of estradiol metabolism may play significant role in development and/or progression of PH.

Radioresistant human lung adenocarcinoma cells that survived multiple fractions of ionizing radiation are sensitive to HSP90 inhibition.

Despite the common usage of radiotherapy for the treatment of NSCLC, outcomes for these cancers when treated with ionizing radiation (IR) are still unsatisfactory. A better understanding of the mechanisms underlying resistance to IR is needed to design approaches to eliminate the radioresistant cells and prevent tumor recurrence and metastases. Using multiple fractions of IR we generated radioresistant cells from T2821 and T2851 human lung adenocarcinoma cells. The radioresistant phenotypes present in T2821/R and T2851/R cells include multiple changes in DNA repair genes and proteins expression, upregulation of EMT markers, alterations of cell cycle distribution, upregulation of PI3K/AKT signaling and elevated production of growth factors, cytokines, important for lung cancer progression, such as IL-6, PDGFB and SDF-1 (CXCL12). In addition to being radioresistant these cells were also found to be resistant to cisplatin.HSP90 is a molecular chaperone involved in stabilization and function of multiple client proteins implicated in NSCLC cell survival and radioresistance. We examined the effect of ganetespib, a novel HSP90 inhibitor, on T2821/R and T2851/R cell survival, migration and radioresistance. Our data indicates that ganetespib has cytotoxic activity against parental T2821 and T2851 cells and radioresistant T2821/R and T2851/R lung tumor cells. Ganetespib does not affect proliferation of normal human lung fibroblasts. Combining IR with ganetespib completely abrogates clonogenic survival of radioresistant cells.Our data show that HSP90 inhibition can potentiate the effect of radiotherapy and eliminate radioresistant and cisplatin -resistant residual cells, thus it may aid in reducing NSCLC tumor recurrence after fractionated radiotherapy.

Ponatinib ameliorates pulmonary fibrosis by suppressing TGF-β1/Smad3 pathway

TGF-β1/Smad3 pathway plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, including lung fibroblasts proliferation and epithelial cell aberrant activation. Ponatinib is a multi-targeted tyrosine-kinase inhibitor. However, whether Ponatinib has anti-fibrotic functions is unknown. In this study, the effects of Ponatinib on TGF-β1-mediated epithelial–mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1), on the apoptosis of human type I alveolar epithelial cells (AT I) in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis was investigated in vivo. Treatment with Ponatinib resulted in a reduction of EMT in A549 cells with a decrease in vimentin and p-Smad3, whereas an increase in E-cadherin. Apoptosis of AT I was attenuated with an increase in the Bcl-2/Bax ratio. HLF-1 proliferation was reduced with a decrease in PDGF-BB and FGF-2 expressions. Treatment with Ponatinib resulted in an amelioration of the BLM-induced pulmonary fibrosis in rats with reductions of the pathological score, collagen deposition, p-Smad3, α-SMA, PDGF-BB and FGF-2 expression. In summary, Ponatinib reversed the EMT, inhibited the apoptosis of AT I, as well as HLF-1 proliferation and prevented pulmonary fibrosis by suppressing the TGF-β1/Smad3 pathway.

Human embryonic lung fibroblasts treated with artesunate exhibit reduced rates of proliferation and human cytomegalovirus infection in vitro.

Cytomegalovirus (CMV) pneumonia is a major cause of death in immunosuppressed patients. Despite the effective treatment with ganciclovir (GCV) and other antiviral agents, the mortality rate remains between 30% to 50%. Recently, the anti-malarial drug artesunate (ART) wasfound to exhibit significant anti-viral activity. Here, we examined the effects of ART on human cytomegalovirus (HCMV) infection and human embryonic lung fibroblast (HELF) proliferation in vitro. HELFs infected with the GFP-expressing Towne-BAC strain of HCMV were divided into three treatment groups: Group I, cells treated with ART for 1.5 h before HCMV inoculation; Group II, cells infected with HCMV that was pre-treated with ART for 1.5 h before HCMV inoculation; Group III, cells that were treated with ART at 1.5 h post-HCMV inoculation. GFP expression was observed daily by fluorescence microscopy, and the number of GFP-positive cells in each experimental group was recorded at 4-5 days post-infection. At 10 days post-infection, the viability of cells in each group was recorded. GCV treatment was used as a control. While no significant effects on cytotoxicity, cell viability, viral infection rates, or antiviral activity were observed upon treatment of Group I or II cells with GCV or low levels of ART, the ART-treated Group III population exhibited significantly reduced rates of infection at drug concentrations higher than 12.5 µM. Similarly, we observed a GCV concentration-dependent reduction in the viral infection rate in Group III cells. Notably, ART-treated, but not GCV-treated, cells also exhibited decreased proliferation. The 50% cytostatic concentrations (CC50) and the half maximal inhibitory concentrations (IC50) of ART and GCV were 54.382 µM and 12.679 µM, and 3.76 M and 14.479 µM, respectively. In addition to its robust antiviral activity, ART inhibits proliferation of HCMV-infected lung fibroblasts, making it a potential next-generation drug for CMV pneumonia treatment and for reducing fibroproliferation and fibrosis in these patients.

Inhibition of connective tissue growth factor attenuates paraquat-induced lung fibrosis in a human MRC-5 cell line.

Chronic exposure to Paraquat (PQ) may result in progressive pulmonary fibrosis and subsequent chronic obstructive pulmonary malfunction. Connective tissue growth factor (CTGF) has been proposed as a key determinant in the development of lung fibrosis. We investigated thus whether knock down of CTGF can prevent human lung fibroblasts (MRC-5) activation and proliferation with the subsequent inhibition of PQ-induced fibrosis. MRC-5 was transfected with CTGF-siRNAs and exposed to different concentrations of PQ. The siRNA-silencing efficacy was evaluated using western blotting analyses, qRT-PCR and flow cytometry. Next, the viability and migration of MRC-5 was determined. MMP-2, MMP-9, and TIMP-1 accumulation were quantified to evaluate the lung fibrosis exposure to PQ. Over expression of CTGF mRNA was observed in human MRC-5 cell as early as 6 h following PQ stimulation. CTGF gene expression in MRC-5 cells was substantially reduced by RNAi, which significantly suppressed the expression of the lung fibrosis markers such as tissue inhibitor of metalloproteinase-2 (TIMP-2), Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) that were stimulated by PQ. Inhibition of CTGF expression suppressed impeded the proliferation and migration ability of MRC-5 cells and resulted in cell-extracellular matrix (ECM) protein accumulation in cells. Our results suggest that CTGF promoted the development of PQ-induced lung fibrosis in collaboration with transforming growth factor β1 (TGFβ1). Furthermore, the observed arresting effects of CTGF knock down during this process suggested that CTGF is the potential target site for preventing PQ-induced pulmonary fibrosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1620-1626, 2016.

Protocatechuic aldehyde ameliorates experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway

An abnormal high mobility group box 1 (HMGB1) activation and a decrease in receptor for advanced glycation end-product (RAGE) play a key role in the pathogenesis of pulmonary fibrosis. Protocatechuic aldehyde (PA) is a naturally occurring compound, which is extracted from the degradation of phenolic acids. However, whether PA has anti-fibrotic functions is unknown. In this study, the effects of PA on the transforming growth factor-β1 (TGF-β1)-mediated epithelial–mesenchymal transition (EMT) in A549 cells, on the apoptosis of human type I alveolar epithelial cells (AT I), on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis in vivo were investigated. PA treatment resulted in a reduction of EMT in A549 cells with a decrease in vimentin and HMGB, an increase of E-cadherin and RAGE, a reduction of HLF-1 proliferation with a decrease of fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor (PDGF). Apoptosis of AT I was attenuated with an increase of RAGE. PA ameliorated BLM-induced pulmonary fibrosis in rats with a reduction of histopathological scores and collagen deposition, and a lower FGF-2, PDGF, α-smooth muscle actin (α-SMA) and HMGB1 expression, whereas higher RAGE was found in BLM-instilled lungs. Through the decrease of HGMB1 and the regulation of RAGE, PA reversed the EMT, inhibited HLF-1 proliferation as well as reduced apoptosis in AT I, and prevented pulmonary fibrosis in vivo. Collectively, our results demonstrate that PA prevents experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway.

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

The stimulating effect of interleukin-33 on proliferation of the primary human lung fibroblasts

Interleukin (IL)-33 is a multifunctional cytokine that belongs to the IL-1 cytokine family and expressed by multiple organs and cell types. Recent studies have showed that IL-33 plays an etiological role in several fibrotic disorders and may be involved in the pathogenesis of chronic respiratory diseases. It has been reported that IL-33–induced cutaneous fibrosis is associated with the increased fibroblast proliferation and altered expression of extracellular matrix-modifying genes. However, the role of IL-33 in regulating of functions of lung fibroblasts remains unclear. In the present study we examined the effect of IL-33 on proliferation of human lung fibroblasts. Five primary lines of normal adult human lung fibroblasts were cultured for 3-7 days in the presence of increasing concentrations of IL-33. We have observed that normal human lung fibroblasts responded in a dose-dependent manner to treatment with recombinant human IL-33 by increasing proliferation rates 1.5- to 2.3 fold compared to the non-stimulated control. The maximum effect of IL-33 on fibroblast proliferation was observed in the cytokine concentrations range from 2 ng/ml to 100 ng/ml. These results suggest that IL-33 may play an important role in the regulation of the human lung fibroblast proliferation. Human lung fibroblasts activated by IL-33 may act as effector cells not only in the pathogenesis of lung diseases, but also in lung remodeling processes.

Transforming growth factor-β1 small interfering RNA inhibits growth of human embryonic lung fibroblast HFL-I cells in vitro and defends against radiation-induced lung injury in vivo.

In the present study, a human transforming growth factor‑β1 (TGF‑β1) small interfering RNA (siRNA) plasmid vector (TGF‑β1‑siRNA) was constructed to investigate its effects on the proliferation and differentiation of human lung fibroblasts in vitro and its interference effects on radiation‑induced lung injury in vivo. Reverse transcription quantitative polymerase chain reaction and enzyme linked immunosorbent assay revealed that the mRNA and protein expression of TGF‑β1 in the HFL‑I cells were inhibited by TGF‑β1‑siRNA and flow cytometry demonstrated a significant increase in apoptosis of the HFL‑I cells. Adult, female, specific‑pathogen‑free C57BL/6 mice were used in the in vivo animal investigations and were randomly divided into the four following groups: control without any treatment, radiation alone, radiation followed by empty vector transfection and radiation followed by TGF‑β1‑siRNA vector transfection. Hematoxylin and eosin and Van‑Gieson staining revealed that certain radiation‑induced histopathological changes of the lung, including inflammation, edema, the density of surface pulmonary interstitial collagen fibers in the alveolar septum, TGF‑β1‑positive reactions in alveolar epithelial cells and pulmonary interstitial macrophages were less marked in the mice transfected with TGF‑β1‑siRNA compared with the mice without transfection or those transfected with empty vectors. The serum levels of TGF‑β1 levels in the irradiated mice increased significantly at four weeks and peaked at eight weeks after radiation, compared with the control. Serum levels of TGF‑β1 in the irradiated mice transfected with TGF‑β1‑siRNA also increased gradually and a significant difference was observed compared with those irradiated without transfection. The mRNA expression levels of TGF‑β1 in the mice transfected with TGF‑β1‑siRNA were markedly lower compared with those of the other radiation groups. The present study suggested that the TGF‑β1‑siRNA vector reduced the activity of TGF‑β1 by downregulating the mRNA expression of TGF‑β1 and thereby effectively suppressing inflammatory reactions and defending against radiation‑induced lung injury.

MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.

Discovery of novel non-carboxylic acid 5-amino-4-cyanopyrazole derivatives as potent and highly selective LPA1R antagonists

High throughput screening (HTS) of our chemical library identified 3-alkylamino-2-aryl-5H-imidazo[1,2,b]pyrazol-7-carbonitrile 1 as a potent antagonist of the LPA1 receptor (LPA1R). Further evaluation of this class of compounds indicated that LPA1R antagonist activity originated from the degradation of the parent molecule in DMSO during the assay conditions. Here, we describe the isolation and characterization of the degradation products and their LPA1R antagonist activity. We further profiled these novel non-carboxylic acid LPA1R antagonists and demonstrated their inhibition of LPA-induced proliferation and contraction of normal human lung fibroblasts (NHLF).

Diallyl disulfide inhibits proliferation and transdifferentiation of lung fibroblasts through induction of cyclooxygenase and synthesis of prostaglandin E₂.

Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) are critically involved in idiopathic pulmonary fibrosis by inducing the proliferation and transdifferentiation of lung fibroblasts. In the present study, we examined the impact of diallyl disulfide (DADS), a garlic-derived compound, on such pathological conditions. DADS showed profound inhibitory effects on the PDGF-BB-induced proliferation of human and mouse lung fibroblasts. DADS also abrogated the TGF-β1-induced expression of α-smooth muscle actin, type I collagen and fibronectin. Following treatment with DADS, the expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E₂ (PGE₂) were found to be markedly enhanced, which in turn led to elevated cAMP levels in lung fibroblasts. Notably, the effect of DADS was largely abolished in the presence of either COX inhibitor indomethacin or siRNA-targeting COX-2, or in the absence of the PGE₂ receptor EP2, supporting an essential role for the COX-2-PGE₂-cAMP autocrine loop. Furthermore, we demonstrated that the upregulated expression of COX-2 was a result of increased level of histone 3 acetylation at COX-2 locus in DADS-treated cells. Together, these results suggest that DADS, by inducing COX-2 expression, may have therapeutic potential in treating lung fibrosis.

Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts

Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.

Thrombin promotes proliferation of human lung fibroblasts via protease activated receptor‐1‐dependent and NF‐κB‐independent pathways

Acute and chronic respiratory diseases are associated with abnormal coagulation regulation and fibrolysis. However, the detailed mechanism by which coagulation regulation and fibrolysis affect the occurrence and development of lung diseases remain to be elucidated. Protease activated receptor-1 (PAR-1), a major high-affinity thrombin receptor, and nuclear factor kappa B (NF-κB), a transcription factor, are involved in cell survival, differentiation, and proliferation. We have investigated the potential mechanism of thrombin-induced fibroblast proliferation and roles of PAR-1 and NF-κB signalling in this process. The effect of thrombin on proliferation of human pulmonary fibroblasts (HPF) was assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. The expression of PAR1 and NF-κB subunit p65 protein was detected by Western blot. Nuclear translocation of p65 was examined by laser scanning confocal microscopy. We show that thrombin significantly increased proliferation of HPF as determined by induction of BrdU-positive incorporation ratio. Induced PAR1 protein expression was also seen in HPF cells treated with thrombin. However, thrombin had no significant effect on expression and translocation of NF-κB p65 in HPF cells. The results indicate that, by increasing protein expression and interacting with PAR1, thrombin promotes HPF proliferation. NF-κB signalling appears to play no role in this process.

Antifibrotic and Anti-inflammatory Activity of the Tyrosine Kinase Inhibitor Nintedanib in Experimental Models of Lung Fibrosis

The tyrosine kinase inhibitor nintedanib (BIBF 1120) is in clinical development for the treatment of idiopathic pulmonary fibrosis. To explore its mode of action, nintedanib was tested in human lung fibroblasts and mouse models of lung fibrosis. Human lung fibroblasts expressing platelet-derived growth factor (PDGF) receptor-α and -β were stimulated with platelet-derived growth factor BB (homodimer) (PDGF-BB). Receptor activation was assessed by autophosphorylation and cell proliferation by bromodeoxyuridine incorporation. Transforming growth factor β (TGFβ)-induced fibroblast to myofibroblast transformation was determined by α-smooth muscle actin (αSMA) mRNA analysis. Lung fibrosis was induced in mice by intratracheal bleomycin or silica particle administration. Nintedanib was administered every day by gavage at 30, 60, or 100 mg/kg. Preventive nintedanib treatment regimen started on the day that bleomycin was administered. Therapeutic treatment regimen started at various times after the induction of lung fibrosis. Bleomycin caused increased macrophages and lymphocytes in the bronchoalveolar lavage (BAL) and elevated interleukin-1β (IL-1β), tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen in lung tissue. Histology revealed chronic inflammation and fibrosis. Silica-induced lung pathology additionally showed elevated BAL neutrophils, keratinocyte chemoattractant (KC) levels, and granuloma formation. Nintedanib inhibited PDGF receptor activation, fibroblast proliferation, and fibroblast to myofibroblast transformation. Nintedanib significantly reduced BAL lymphocytes and neutrophils but not macrophages. Furthermore, interleukin-1β, KC, TIMP-1, and lung collagen were significantly reduced. Histologic analysis showed significantly diminished lung inflammation, granuloma formation, and fibrosis. The therapeutic effect was dependent on treatment start and duration. Nintedanib inhibited receptor tyrosine kinase activation and the proliferation and transformation of human lung fibroblasts and showed antifibrotic and anti-inflammatory activity in two animal models of pulmonary fibrosis. These results suggest that nintedanib may impact the progressive course of fibrotic lung diseases such as idiopathic pulmonary fibrosis.

Identification of Two Novel Anti-Fibrotic Benzopyran Compounds Produced by Engineered Strains Derived from Streptomyces xiamenensis M1-94P that Originated from Deep-Sea Sediments

The benzopyran compound obtained by cultivating a mangrove-derived strain, Streptomyces xiamenensis strain 318, shows multiple biological effects, including anti-fibrotic and anti-hypertrophic scar properties. To increase the diversity in the structures of the available benzopyrans, by means of biosynthesis, the strain was screened for spontaneous rifampicin resistance (Rif), and a mutated rpsL gene to confer streptomycin resistance (Str), was introduced into the S. xiamenensis strain M1-94P that originated from deep-sea sediments. Two new benzopyran derivatives, named xiamenmycin C (1) and D (2), were isolated from the crude extracts of a selected Str-Rif double mutant (M6) of M1-94P. The structures of 1 and 2 were identified by analyzing extensive spectroscopic data. Compounds 1 and 2 both inhibit the proliferation of human lung fibroblasts (WI26), and 1 exhibits better anti-fibrotic activity than xiamenmycin. Our study presents the novel bioactive compounds isolated from S. xiamenensis mutant strain M6 constructed by ribosome engineering, which could be a useful approach in the discovery of new anti-fibrotic compounds.

Inhibition of TRPM7 channels prevents proliferation and differentiation of human lung fibroblasts

Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in human lung fibroblast (MRC5) proliferation and differentiation induced by transforming growth factor β1 (TGF-β1) in vitro. We determined the expression of TRPM7 in MRC5 cells in response to TGF-β1 treatment in vitro. Chemical inhibitors (Gd(3+) and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in MRC5 cell proliferation and differentiation. The phosphorylation of Akt was determined by Western blotting. The expression of TRPM7 was significantly potentiated in response to TGF-β1. Co-incubation of MRC5 cells with Gd(3+), 2-APB or TRPM7-siRNA decreased cell proliferation and differentiation. Furthermore, we found that suppression of TRPM7 channels also reduced the p-Akt in MRC5 cells induced by TGF-β1. We conclude that suppression of TRPM7 channels may decrease fibroblast proliferation and differentiation stimulated by TGF-β1 in vitro and this is associated with Akt phosphorylation.