Proliferation Of Pulmonary Fibroblasts Research Articles

Kangfuxin Oral Liquid Attenuates Bleomycin-Induced Pulmonary Fibrosis via the TGF-β1/Smad Pathway.

Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease with a poor prognosis characterized by transforming growth factor (TGF)-β-induced proliferation, migration, and differentiation of fibroblasts, resulting in excessive extracellular matrix (ECM) deposition. Whether Kangfuxin oral liquid (KFXOL) has a protective function in pulmonary fibrosis is largely unknown. The goal of this study was to investigate the potential efficacy of KFXOL, as well as the underlying mechanism by which KFXOL regulates pulmonary fibrosis in vivo and in vitro. We found that KFXOL dramatically attenuated intratracheal bleomycin (BLM)-induced pulmonary fibrosis in terms of both severe alveolar architecture destruction and collagen deposition. KFXOL treatment significantly inhibited the proliferation, migration, and differentiation of pulmonary fibroblasts following activation using BLM/TGF-β1 and normalized the expression of ECM deposition-related proteins, including matrix metalloproteinase (MMP)-1, MMP-9, and tissue inhibitor of metalloproteinases 1. These effects were mediated via the inhibition of TGF-β1 and phosphorylated Smad2/3 activation in vivo. Taken together, our data suggest that KFXOL attenuates the development of pulmonary fibrosis via the TGF-β1/Smad signaling pathway and thus has potential utility in the treatment of pulmonary fibrosis.

RNA-binding protein SFPQ cooperates with HDAC1 to suppress CD40 transcription in pulmonary adventitial fibroblasts.

Pulmonary artery adventitial fibroblasts, the most abundant cellular constituent of adventitia, are often the first to be activated and reprogrammed to then influence the tone and structure of the vessel wall in pulmonary arterial hypertension (PAH). Our previous study found that interruption of CD40 ligand (CD40L)-CD40 signaling improves the efficacy of transplanted endothelial progenitor cells in monocrotaline induced-PAH. However, whether CD40L-CD40 signaling is involved in the activation of adventitial fibroblasts in PAH and whether Drosophila behavior human splicing (DBHS) protein family members have any roles during adventitial fibroblasts activation are completely unclear. Here, we show that soluble CD40L (sCD40L) stimulation progressively increases pro-inflammatory activity, proliferation, and migration of pulmonary adventitial fibroblasts. Besides, sCD40L stimulation decreases splicing factor proline- and glutamine-rich protein (SFPQ) protein (one member of DBHS protein family) expression, while SFPQ overexpression suppresses sCD40L stimulation-induced proliferation and migration of pulmonary adventitial fibroblasts by repressing CD40 transcription. Moreover, ChIP assays found that sCD40L stimulation promotes histone H3 tri-methylation at lysine 4 (H3K4me3), H3K36me3, and H3K27 acetylation (H3K27ac) modifications on CD40 promoter region in pulmonary adventitial fibroblasts, while SFPQ overexpression decreases H3K36me3 modification and increases H3K36ac on CD40 promoter region by interacting with histone deacetylase-1 (HDAC1) to inhibit CD40 transcription. This in-depth study shows that CD40L-CD40 signaling promotes activation of pulmonary adventitial fibroblasts by increasing proliferation, migration, and pro-inflammatory activity of adventitial fibroblasts, and SFPQ could inhibit CD40 transcription though switching H3K36me3 to H3K36ac modifications on its promoter by interacting with HDAC1. This study, first, uncovers the roles of SFPQ on CD40L-CD40 signaling-mediated activation of pulmonary adventitial fibroblasts.

Molecular genetic framework underlying pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare, progressive disorder typified by occlusion of the pulmonary arterioles owing to endothelial dysfunction and uncontrolled proliferation of pulmonary artery smooth muscle cells and fibroblasts. Vascular occlusion can lead to increased pressure in the pulmonary arteries, often resulting in right ventricular failure with shortness of breath and syncope. Since the identification of BMPR2, which encodes a receptor in the transforming growth factor-β superfamily, the development of high-throughput sequencing approaches to identify novel causal genes has substantially advanced our understanding of the molecular genetics of PAH. In the past 6 years, additional pathways involved in PAH susceptibility have been described through the identification of deleterious genetic variants in potassium channels (KCNK3 and ABCC8) and transcription factors (TBX4 and SOX17), among others. Although familial PAH most often has an autosomal-dominant pattern of inheritance, cases of incomplete penetrance and evidence of genetic heterogeneity support a model of PAH as a Mendelian disorder with complex disease features. In this Review, we outline the latest advances in the detection of rare and common genetic variants underlying PAH susceptibility and disease progression. These findings have clinical implications for lung vascular function and can help to identify mechanistic pathways amenable to pharmacological intervention.

MicroRNA-328 in exosomes derived from M2 macrophages exerts a promotive effect on the progression of pulmonary fibrosis via FAM13A in a rat model

Currently, exosome-enclosed microRNAs (miRs) in exhaled breath have potential for biomarker discovery in patients with pulmonary diseases. This study was performed to investigate the roles of M2 macrophage-derived exosomes expressing miR-328 in pulmonary fibrosis (PF). Microarray-based analysis was used to screen differentially expressed genes (DEGs) and regulatory miRs in PF. The miR-target relationship between FAM13A and miR-328 was confirmed. The expression of FAM13A and miR-328 was measured in PF rats, and gain- and loss-of-function assays were conducted to determine the regulatory effects of FAM13A and miR-328 on PF. In addition, exosomes derived from M2 macrophages were isolated and then cocultured with pulmonary interstitial fibroblasts to identify the role of these exosomes in PF. Furthermore, the effects of M2 macrophage-derived exosomes overexpressing miR-328 on pulmonary fibroblast proliferation and the progression of PF were assessed in vivo. miR-328 might perform a vital function in PF by regulating FAM13A. FAM13A expression was downregulated while miR-328 expression was upregulated in rats with PF, and a miR-target relationship between miR-328 and FAM13A was observed. Additionally, miR-328 overexpression and FAM13A silencing each were suggested to promote pulmonary interstitial fibroblast proliferation and the expression of Collagen 1A, Collagen 3A and α-SMA. Then, in vitro experiments demonstrated that M2 macrophage-derived exosomes overexpressing miR-328 contributed to enhanced pulmonary interstitial fibroblast proliferation and promoted PF. Furthermore, in vivo experiments confirmed the promotive effects of M2 macrophage-derived exosomes overexpressing miR-328 on the progression of PF. Collectively, the results showed that M2 macrophage-derived exosomes overexpressing miR-328 aggravate PF through the regulation of FAM13A.

CD4+CXCR4+ T cells as a novel prognostic biomarker in patients with idiopathic inflammatory myopathy-associated interstitial lung disease

BACKGROUD There is an unmet need for the development of new biomarkers for idiopathic inflammatory myopathy-associated interstitial lung disease (IIM-ILD). METHODS Peripheral CD4+CXCR4+ T cells, stromal cell-derived factor-1 and Krebs von den Lungen-6 were measured in patients with IIM-ILD (n = 85) and controls. The relation to pulmonary functions, high-resolution CT scores, specific clinical phenotypes and survival was analysed. Cytokine-expression profiling of these CD4+CXCR4+ T cells and their co-culture with pulmonary fibroblasts were conducted. RESULTS The peripheral percentages of CD4+CXCR4+ T cells were significantly elevated in IIM-ILD patients, and correlated with high-resolution CT score (r = 0.7136, P < 0.0001) and pulmonary function impairments, such as percentage of forced volume vital capacity (r = -0.4734, P = 0.0005). They were associated with anti-melanoma differentiation-associated gene 5 autoantibodies and the amyopathic DM phenotype. In IIM-ILD, peripheral percentages of CD4+CXCR4+ T cells ⩾30% revealed a 6-month mortality as high as 47%. These CD4+CXCR4+ T cells express high levels of IL-21 and IL-6. In vitro blockade of IL-21 signalling by neutralization of IL-21 or Janus kinase inhibitor could abolished the fibroblast proliferation. CONCLUSION Overall, peripheral CD4+CXCR4+ T cells appear to be a potentially valuable novel biomarker associated with the severity and prognosis of IIM-ILD. They promote pulmonary fibroblast proliferation via IL-21, which may herald future targeted treatments for this severe disease.

CD4+CXCR4+ T cells as a novel prognostic biomarker in patients with idiopathic inflammatory myopathy-associated interstitial lung disease.

There is an unmet need for the development of new biomarkers for idiopathic inflammatory myopathy-associated interstitial lung disease (IIM-ILD). Peripheral CD4+CXCR4+ T cells, stromal cell-derived factor-1 and Krebs von den Lungen-6 were measured in patients with IIM-ILD (n = 85) and controls. The relation to pulmonary functions, high-resolution CT scores, specific clinical phenotypes and survival was analysed. Cytokine-expression profiling of these CD4+CXCR4+ T cells and their co-culture with pulmonary fibroblasts were conducted. The peripheral percentages of CD4+CXCR4+ T cells were significantly elevated in IIM-ILD patients, and correlated with high-resolution CT score (r = 0.7136, P < 0.0001) and pulmonary function impairments, such as percentage of forced volume vital capacity (r = -0.4734, P = 0.0005). They were associated with anti-melanoma differentiation-associated gene 5 autoantibodies and the amyopathic DM phenotype. In IIM-ILD, peripheral percentages of CD4+CXCR4+ T cells ⩾30% revealed a 6-month mortality as high as 47%. These CD4+CXCR4+ T cells express high levels of IL-21 and IL-6. In vitro blockade of IL-21 signalling by neutralization of IL-21 or Janus kinase inhibitor could abolished the fibroblast proliferation. Overall, peripheral CD4+CXCR4+ T cells appear to be a potentially valuable novel biomarker associated with the severity and prognosis of IIM-ILD. They promote pulmonary fibroblast proliferation via IL-21, which may herald future targeted treatments for this severe disease.

The KCa3.1 blocker TRAM-34 inhibits proliferation of fibroblasts in paraquat-induced pulmonary fibrosis

KCa3.1, a Ca2+-activated K+ channel, plays an important role in modulating calcium signaling and maintaining membrane potential during cell activation. It has been reported to promote fibroblast function in many fibrotic diseases. However, the role of KCa3.1 in the pathophysiology of pulmonary fibrosis after paraquat (PQ) poisoning has not been studied. A rat model of PQ poisoning was used. After treatment with TRAM-34, which is a highly selective KCa3.1 blocker, the expression of KCa3.1, TGF-β1 and α-SMA were evaluated via Western blot, histology and other assays. Bromodeoxyuridine (BrdU) marking and MTT assay were used to measure primary rat pulmonary fibroblast proliferation. The results showed that KCa3.1 expression was elevated after PQ poisoning. Blockade of KCa3.1 alleviated PQ-induced pulmonary inflammation and fibrosis. Blockade of KCa3.1 also attenuated the level of collagen I and α-SMA and the proliferation of fibroblasts. However, TGF-β1 expression remained unaffected by blockade of KCa3.1 in rat lung tissues after PQ poisoning. The present study suggests that KCa3.1 expression increased and might promote pulmonary fibroblast proliferation in PQ-induced pulmonary fibrosis. In addition, we confirmed that TRAM-34 attenuates proliferation and collagen secretion of fibroblasts. Our findings indicated that TRAM-34 might inhibit PQ-induced proliferation of pulmonary fibroblasts and prevent progression of lung fibrosis.

Cell cycle regulation of pulmonary fibroblasts proliferation in neonate rats induced by transforming growth factor- β1

Objective To investigate the effect of transforming growth factor(TGF)-β1 stimulation on the proliferation of lung fibroblasts and the pathogenesis of bronchopulmonary dysplasia(BPD). Methods The lung fibroblasts of newborn rats were isolated, purified and identified on the 1st day after birth.The primary lung fibroblasts were stimulated by TGF-β1, and the protein expression of cyclin-dependent kinase-2(CDK2) and p27 were detected by immunohistochemistry and Western blotting.Real-time fluorescence quantitative polymerase chain reaction(PCR) was used to detect the mRNA expression of CDK2 and p27.CCK8 kit was used to detect cell proliferation, and cell cycle was detected by flow cytometry. Results The expression of CDK2 protein and mRNA in primary fibroblasts was increased after TGF-β1 stimulation in lung fibroblasts of newborn rats, accompanied by the decrease of p27 protein and mRNA.CCK8 showed that the value of A increased significantly, proliferation significantly increased the proportion of S phase cells at the same time, G0/G1 phase decreased.The percentage of S phase cells after stimulation with 0 μg/L, 10 μg/L TGF-β1 was 4.63%, 67.09%, respectively.The percentage of G0/G1 phase was 83.67% and 67.09%, respectively.The percentage of S phase cells was 7.64% and 9.11% respectively after stimulated with 10 μg/L TGF-β1 for 12 h and 48 h, respectively.The percentage of G0/G1 phase was 73.99% and 59.81% respectively.With the increase of TGF-β1 levels (0 μg/L, 5 μg/L and 10 μg/L), CDK2 protein and mRNA expression further increased; and the same level of TGF-β1 stimulation, stimulated for 12, 24 and 48 h, the expression of CDK2 protein and mRNA increased further, and increased significantly at 48 h (P<0.05 and <0.01 respectively). The levels of p27 protein and mRNA decreased further(all P<0.01). Conclusions TGF-β1 can up-regulate the expression of CDK2/p27 in primary lung fibroblasts of neonatal rats, accelerating cell cycle progression and over-proliferation of fibroblasts. Key words: Bronchopulmonary dysplasia; Transforming growth factor-β1; p27; Cyclin-dependent kinase-2

Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3–6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

Hypoxia induces pulmonary arterial fibroblast proliferation, migration, differentiation and vascular remodeling via the PI3K/Akt/p70S6K signaling pathway.

The present study was designed to examine whether hypoxia induces the proliferation, migration and differentiation of pulmonary arterial fibroblasts (PAFs) via the PI3K/Akt/p70S6K signaling pathway. PAFs were subjected to normoxia (21% O2) or hypoxia (1% O2). The proliferation, migration, differentiation and cellular p110α, p-Akt, and p-p70S6K expression levels of the PAFs were examined in vitro. In addition, rats were maintained under hypoxic conditions, and the right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and right ventricular weight/body weight ratio (RV/BW) were examined. The expression levels of p110α, p-Akt, p70S6K, fibronectin and α-SMA in the rat pulmonary vessels were also examined. Hypoxia significantly elevated the proliferation, migration and differentiation of rat PAFs. It also strongly elevated the expression of p110α, p-Akt and p-p70S6K in PAFs in vitro. NVP-BEZ235 was revealed to significantly reduce the hypoxia-induced proliferation, migration and differentiation. In vivo experiments demonstrated that hypoxia significantly induced the elevation of RVSP, RVHI, RV/BW, medial thickening, adventitious thickening, and fibronectin and collagen deposition around pulmonary artery walls. The expression of p110α, p-Akt and p70S6K was evident in the pulmonary arteries of the hypoxic rats. NVP-BEZ235 significantly reduced the hypoxia-induced hypoxic pulmonary vascular remodeling, as well as fibronectin and collagen deposition in the pulmonary arteries. Therefore, hypoxia was demonstrated to induce the proliferation, migration and differentiation of PAFs and the hypoxic pulmonary vascular remodeling of rats via the PI3K/Akt/p70S6K signaling pathway.

Shikonin suppresses pulmonary fibroblasts proliferation and activation by regulating Akt and p38 MAPK signaling pathways

Fibroblast is believed to be the primary effector in idiopathic pulmonary fibrosis (IPF), a progressive lung disorder characterized by aberrant tissue remodeling and the formation of fibroblastic foci. Due to the complicated etiology and mechanism, there are few effective drugs for this fatal disease.Shikonin (SHI), which is the major ingredient isolated from the plant Lithospermum Erythrorhizon, has long been used as traditional medicine for many diseases including inflammation and cancer. The roles of SHI in attenuating skin scar and renal fibrosis by reducing TGFβ1-stimulated fibroblast activation are also reported. But whether SHI works on IPF which exhibits both inflammatory and carcinoma-like features remains unknown.In this study, using isolated pulmonary fibroblasts, we demonstrated that SHI inhibited the proliferation, migration of fibroblasts, enhanced cell apoptosis and led to cell cycle arrest at G1 and G2/M phase. Moreover, SHI reduced the production of α-SMA, fibronectin, collagen I and III in response to TGF-β induction in pulmonary fibroblasts, and all of these gene production is the key component of extracellular matrix for tissue remodeling for IPF. The phosphorylation of Akt was down-regulated, p53 increased, the mRNA levels of p21 and p27 enhanced after SHI treatments. The phosphorylation of both p38 MAPK and Akt stimulated by TGF-β was reduced after SHI treatments.Collectively, these data indicate that SHI has a strong cytotoxicity in pulmonary fibroblast via inhibiting Akt activation signaling pathway, and attenuates TGF-β induced extracellular matrix genes production in pulmonary fibroblasts via modulating the activities of p38 MAPK and Akt. SHI might serve as a therapeutically candidate for IPF patients.

Galectin-3 mediates pulmonary vascular remodeling in hypoxia-induced pulmonary arterial hypertension

Pulmonary vascular adventitia serves as a key regulator of pulmonary vascular remodeling in the pathogenesis of pulmonary arterial hypertension (PAH). Excessive proliferation and differentiation of pulmonary adventitial fibroblasts (PAFs) are proven to be crucial in the pathogenesis of PAH. Galectin-3 (Gal-3) is known as a key fibroblasts activating factor which is involved in the fibrogenesis of several diseases, such as pulmonary fibrosis, vascular fibrosis, and heart failure. Therefore, we seek to investigate the potential role of Gal-3 in regulating PAF cells in the pathogenesis of PAH. Gal-3 plasma concentration was significantly higher in PAH patients. Gal-3 was upregulated in pulmonary artery adventitia of hypoxia-induced PAH rats. Inhibition of Gal-3 with N-Acetyl-D-lactosamine (N-Lac) ameliorated PAH and pulmonary vascular remodeling. Gal-3 can stimulate the proliferation, differentiation, and collagen synthesis of PAFs, which was reversed by N-Lac. Transforming growth factor β1 increased Gal-3 expression in PAFs, whereas N-Lac significantly suppressed transforming growth factor β1–induced proliferation, differentiation, and collagen synthesis of PAFs. Gal-3 serves as a critical regulator in the pathogenesis of PAH by regulating the proliferation, differentiation, and extracellular matrix deposition synthesis of PAFs. Inhibition of Gal-3 may represent a novel therapeutic target for PAH treatment.

The Role of miR-497/EIF3A Axis in TGFβ1-Induced Epithelial-Mesenchymal Transition and Extracellular Matrix in Rat Alveolar Epithelial Cells and Pulmonary Fibroblasts.

Multi-cause-induced interstitial lung disease, particularly pulmonary fibrosis, is a serious clinical concern. Fibroblasts have been suggested to have a major role, with it recently being revealed that some of these fibroblasts are derived from alveolar epithelial cells through epithelial-mesenchymal transition (EMT). Eukaryotic translation initiation factor 3 subunit A (EIF3A) is a protein that in humans is encoded by the EIF3A gene, and has been suggested to play roles in regulating translation of a subset of mRNAs and in regulating cell cycle progression and cell proliferation. In the present study, we chose a well-known TGFβ1-induced EMT model in alveolar epithelial cells to investigate the functional role of EIF3A. TGFβ1-induced EIF3A expression and EMT process in alveolar epithelial cells, after EIF3A knockdown, the EMT process could be partially reversed. Online tools and luciferase assays showed that miR-497 could inhibit EIF3A expression by directly binding to the 3'UTR of EIF3A. Ectopic miR-497 expression partially reversed TGFβ1-induced EMT in alveolar epithelial cells. In addition, miR-497 could suppress TGFβ1-induced pulmonary fibroblast proliferation and EIF3A, Collagen I and α-SMA protein levels. Taken together, EIF3A could promote TGFβ1-induced EMT in alveolar epithelial cells; miR-497 suppressed TGFβ1-induced EMT in alveolar epithelial cells TGFβ1-induced excessive proliferation and ECM in pulmonary fibroblast through inhibiting EIF3A by targeting. MiR-497/EIF3A axis shows the potential to be effective in the treatment of pulmonary fibrosis. J. Cell. Biochem. 118: 3401-3408, 2017. © 2017 Wiley Periodicals, Inc.

Effects of calcitonin gene-related peptide on eIF3a and p27 expression in bleomycin-induced pulmonary fibrosis of rats

To observe the effects of calcitonin gene-related peptide (CGRP) on eukaryotic translation initiation factor 3a (eIF3a) and p27 expression in bleomycin-induced pulmonary fibrosis of rats and its possible mechanism. Twenty-four male SD rats weighing 180~220 g were randomly divided into three groups (n=8):control group, bleomycin group, bleomycin plus capsaicin group. Bleomycin (5 mg/kg) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg/kg·d) by subcutaneous injections 4 days before to deplete endogenous CGRP. At the end of experiments, blood samples were collected from carotid artery to determinate the plasma levels of CGRP by ELISA. The cells were divided into 6 groups as follows:control group, transforming growth factor-β1 (TGF-β1) group, +CGRP (1, 10, 100 nmol/L) group, +CGRP 100 nmol/L and CGRP8-37 1 μmol/L group respectively(n=9). TGF-β1 (5 ng/ml) stimulated proliferation of pulmonary fibroblasts and proliferation was measured by BrdU marking. The expression levels of eIF3a, p27, α-smooth muscle actin (α-SMA) and collagen Ⅰ were detected by immunohistochemisty, real-time PCR or Western blot. The expressions of eIF3a, α-SMA, and collagen I were increased and the expression of p27 was decreasing in pulmonary fibrosis rats induced by bleomycin. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts and the expressions of α-SMA, collagen I and eIF3a, and upregulated the expression of p27. All these effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the eIF3a/p27 signaling pathway.

KCa3.1 channel-blockade reduces both human and ovine pulmonary fibroblast proliferation in vitro

KCa3.1 channel-blockade reduces both human and ovine pulmonary fibroblast proliferation in vitro

Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis.

We have found that eIF3a plays an important role in bleomycin-induced pulmonary fibrosis, and up-regulation of eIF3a induced by TGF-β1 is mediated via the ERK1/2 pathway. Whether ERK1/2 - eIF3a signal pathway is involved in calcitonin gene-related peptide (CGRP)-mediated pathogenesis of bleomycin-induced pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. Sensory CGRP depletion by capsaicin exacerbated bleomycin-induced pulmonary fibrosis in rats, as shown by a significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, accompanied with increased expression of TGF-β1, eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts concomitantly with decreased expression of eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. These effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the ERK1/2 - eIF3a signaling pathway.

Sodium tanshinone IIA sulfonate suppresses pulmonary fibroblast proliferation and activation induced by silica: role of the Nrf2/Trx pathway.

Alveolar macrophages are believed to induce oxidative stress via reactive oxygen species (ROS) when silica particles are inhaled. This process can contribute to the pathogenesis of silicosis, but the mechanism is unclear. A traditional Chinese herbal derivative, sodium tanshinone IIA sulfonate (STS), displays significant antioxidant effects. Here, we determine whether STS can attenuate the oxidative stress induced by silica. Traditionally, studies on the toxic effects of silica have focused on monocultures of macrophages or fibroblasts. A coculture model of macrophages (Raw 264.7) and pulmonary fibroblasts (MRC-5) was used in this study to mimic a more in vivo-like environment. We investigated the protective effects of STS on the abnormal proliferation of MRC-5 fibroblasts in an in vitro model. The results showed that fibroblast viability increased with the accumulation of intracellular ROS induced by cocultured Raw 264.7 cells after silica exposure. Treatment with STS markedly ameliorated the silica-induced cell proliferation and oxidative stress. Western blotting and immunofluorescence analysis of the Nrf2 and thioredoxin (Trx) system were conducted, and the results confirmed that treatment with STS enhanced nuclear Nrf2 accumulation and mediated antioxidant Trx system expression. These findings suggest that silica exposure might induce some level of oxidative stress in fibroblasts and that STS might augment antioxidant activities via up-regulation of the Nrf2 and Trx system pathways in MRC-5 cells in vitro.

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.&lt; 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&lt;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.

44 ASK-1 inhibition prevents hypoxia-induced pulmonary artery fibroblast proliferation and migration in anin vitrocellular model of pulmonary hypertension

Rationale Pulmonary arterial hypertension (PAH) involves progressive remodelling of the pulmonary arteries, increasing pulmonary vascular resistance leading to right ventricular dysfunction. The remodelling is due to increased pulmonary adventitial fibroblast proliferation, migration, and release of cytokines. These fibroblast responses involve members of the Mitogen Activated Protein Kinases (MAPK) signalling pathway. Apoptosis Signal-regulating Kinase-1 (ASK1) is an apical MAPK, which undergoes auto-phosphorylation and activation in response to oxidative stress, activating downstream p38 MAPK and c-Jun N-terminal kinase (JNK). The work presented here investigates the possible cellular mechanisms and downstream targets of ASK1 in the pulmonary artery fibroblast (PAF) in a model of PAH. Methodology PAF were obtained from adult Sprague Dawley rats. Hypoxia (5% O2) was used as an in vitro model of PAH. Proliferation was determined using [3H] thymidine incorporation. Migration was determined by scratch assay. Targets of ASK1 were determined by Western Blotting. ASK1 inhibition was achieved by the addition of a selective, small molecule inhibitor (GS-444217). Results Hypoxia increased PAF proliferation and migration; both were significantly reduced by the addition of 1 µM GS-444217. Hypoxia increased phosphorylation of both ASK1 and p38 MAPK, and increased Hypoxia-Inducible Factor 1 alpha (HIF1a) protein abundance; 1 µM GS-444217 attenuated these effects. ASK1 inhibition reduced hypoxia-induced expression of key mediators of vascular remodelling, tissue Inhibitor of Metaloproteinase-1 (TIMP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and endothelin-1 (ET-1). Conclusions ASK1 inhibitor (GS-444217) reduced hypoxia-induced proliferation and migration of PAF, and reduced disease-associated signalling pathways. ASK1 inhibition may be a novel target for preventing maladaptive vascular remodelling.

The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension.

The p38 mitogen-activated protein kinase (MAPK) system is increasingly recognized as an important inflammatory pathway in systemic vascular disease but its role in pulmonary vascular disease is unclear. Previous in vitro studies suggest p38 MAPKα is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodeling (PVremod). In this study the role of the p38 MAPK pathway was investigated in both in vitro and in vivo models of pulmonary hypertension and human disease. Pharmacological inhibition of p38 MAPKα in both chronic hypoxic and monocrotaline rodent models of pulmonary hypertension prevented and reversed the pulmonary hypertensive phenotype. Furthermore, with the use of a novel and clinically available p38 MAPKα antagonist, reversal of pulmonary hypertension was obtained in both experimental models. Increased expression of phosphorylated p38 MAPK and p38 MAPKα was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in the PVremod A reduction of IL-6 levels in serum and lung tissue was found in the drug-treated animals, suggesting a potential mechanism for this reversal in PVremod. This study suggests that the p38 MAPK and the α-isoform plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6. Selective inhibition of this pathway may provide a novel therapeutic approach that targets both remodeling and inflammatory pathways in pulmonary vascular disease.