Mitofusion 2 Overexpression Decreased Proliferation of Human Embryonic Lung Fibroblasts in Acute Respiratory Distress Syndrome through Inhibiting RAS-RAF-1-ERK1/2 Pathway

Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fibroproliferation is an essential mechanism in ARDS.Mitofusion2(Mfn2)overexpression plays a role in inhibiting cell proliferation.However,the role and potential mechanism of Mfn2 on the proliferation of fibroblasts is still unknown.In this study,we aimed at exploring the effect of Mfn2 on the human embryonic lung fibroblasts(HELF)and discussed its related mechanism.The HELF were treated with the Mfn2 overexpressing lentivirus(adv-Mfn2).The cell cycle was detected by flow cytometry.MTT,PCR and Western blotting were used to investigate the effect of Mfn2 on the proliferation of the HELF,collagen expression,the RAS-RAF-1-ERK1/2 pathway and the expression of cycle-related proteins(p21,p27,Rb,Raf-1,p-Raf-1,Erk1/2 and p-Erk1/2).The co-immunoprecipitation assay was used to explore the interaction between Mfn2 and Ras.The results showed that the overexpression of Mfn2 inhibited the proliferation of the HELF and induced the cell cycle arrest at the G0/G1 phase.Meanwhile,Mfn2 also inhibited the expression of collagen I,p-Erk and p-Raf-1.In addition,an interaction between Mfn2 and Ras existed in the HELF.This study suggests that the overexpression of Mfn2 can decrease the proliferation of HELF in ARDS,which was associated with the inhibition of the RAS-RAF-1-ERK1/2 pathway.The results may offer a potential therapeutic intervention for patients with ARDS.

Role of Insulin-like Growth Factor II Receptor in Transdifferentiation of Free Silica-induced Primary Rat Lung Fibroblasts

Objective To study the role of insulin-like growth factor II receptor in free silica-induced transdifferentiation of primary rat lung fibroblasts Methods Rat lung fibroblasts and rat alveolar macrophages were cultured. A transdifferentiation model of primary rat lung fibroblasts was induced by free silica. Levels of a-SMA protein, IGF-liR protein and mRNA were measured by immunocytochemistry, Western blot and RT-PCR, respectively. Lung fibroblasts were treated with Wortmannin. Results The expression levels of a-SMA concentration and decreased after Wortmann and IGF-IIR increased with the increasing free silica n was used. Conclusion The IGF-IIR plays an important role in free silica-induced transdifferentiation of primary rat lung fibroblasts.

Retinoic Aacid Diminished the Expression of MMP-2 in Hyperoxia-exposed Premature Rat Lung Fibroblasts through Regulating Mitogen-activated Protein Kinases

This study examined the effects of retinoic acid （RA）, PD98059, SP600125 and SB203580 on the hyperoxia-induced expression and regulation of matrix metalloproteinase-2 （MMP-2） and metalloproteinase-2 （TIMP-2） in premature rat lung fibroblasts （LFs）. LFs were exposed to hyperoxia or room air for 12 h in the presence of RA and the kinase inhibitors PD98059 （ERK1/2）, SP600125 （JNK1/2） and SB203580 （p38） respectively. The expression levels of MMP-2 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. MMP-2 activity was measured by zymography. The amount of p-ERK1/2, REK1/2, p-JNK1/2, JNK1/2, p-p38 and p38 was determined by Western blotting. The results showed that： （1） PD98059, SP600125 and SB203580 significantly inhibited p-ERK1/2, p-JNK1/2 and p-p38 respectively in LFs; （2） The expression of MMP-2 mRNA in LFs exposed to hyperoxia was decreased after treatment with RA, SP600125 and SB203580 respectively （P0.01 or 0.05）, but did not change after treatment with PD98059 （P0.05）. Meanwhile, RA, PD98059, SP600125 and SB203580 had no effect on the expression of TIMP-2 mRNA in LFs exposed to room air or hyperoxia （P0.05）; （3） The expression of pro- and active MMP-2 experienced no change after treatment with RA or SP600125 in LFs exposed to room air （P0.05）, but decreased remarkably after hyperoxia （P0.01 or 0.05）. SB203580 inhibited the expression of pro- and active MMP-2 either in room air or under hyperoxia （P0.01）. PD98059 exerted no effect on the expression of pro- and active MMP-2 （P0.05）. It was suggested that RA had a protective effect on hyperoxia-induced lung injury by down-regulating the expression of MMP-2 through decreasing the JNK and p38 activation in hyperoxia.

Macrophage exosomes transfer angiotensin Ⅱ type 1 receptor to lung fibroblasts mediating bleomycin-induced pulmonary fibrosis

Background:Macrophages are involved in the pathogenesis of idiopathic pulmonary fibrosis,partially by activating lung fibroblasts.However,how macrophages communicate with lung fibroblasts is largely unexplored.Exosomes can mediate intercellular communication,whereas its role in lung fibrogenesis is unclear.Here we aim to investigate whether exosomes can mediate the crosstalk between macrophages and lung fibroblasts and subsequently induce fibrosis.Methods:In vivo,bleomycin(BLM)-induced lung fibrosis model was established and macrophages infiltration was examined.The effects of GW4869,an exosomes inhibitor,on lung fibrosis were assessed.Moreover,macrophage exosomes were injected into mice to observe its pro-fibrotic effects.In vitro,exosomes derived from angiotensin Ⅱ(Ang Ⅱ)-stimulated macrophages were collected.Then,lung fibroblasts were treated with the exosomes.Twenty-four hours later,protein levels ofα-collagen I,angiotensin Ⅱ type 1 receptor(AT1R),transforming growth factor-β(TGF-β),and phospho-Smad2/3(p-Smad2/3)in lung fibroblasts were examined.The Student's t test or analysis of variance were used for statistical analysis.Results:In vivo,BLM-treated mice showed enhanced infiltration of macrophages,increased fibrotic alterations,and higher levels of Ang Ⅱ and AT1R.GW4869 attenuated BLM-induced pulmonary fibrosis.Mice with exosomes injection showed fibrotic features with higher levels of Ang Ⅱ and AT1R,which was reversed by irbesartan.In vitro,we found that macrophages secreted a great number of exosomes.The exosomes were taken by fibroblasts and resulted in higher levels of AT1R(0.22±0.02 vs.0.07±0.02,t=8.66,P=0.001),TGF-β(0.54±0.05 vs.0.09±0.06,t=10.00,P<0.001),p-Smad2/3(0.58±0.06 vs.0.07±0.03,t=12.86,P<0.001)andα-collagen I(0.27±0.02 vs.0.16±0.01,t=7.01,P=0.002),and increased Ang Ⅱ secretion(62.27±7.32 vs.9.56±1.68,t=12.16,P<0.001).Interestingly,Ang Ⅱ increased the number of macrophage exosomes,and the protein levels of Alix(1.45±0.15 vs.1.00±0.10,t=4.32,P=0.012),AT1R(4.05±0.64 vs.1.00±0.09,t=8.17,P=0.001),and glyceraldehyde-3-phosphate dehydrogenase(2.13±0.36 vs.1.00±0.10,t=5.28,P=0.006)were increased in exosomes secreted by the same number of macrophages,indicating a positive loop between Ang Ⅱ and exosomes production.Conclusions:Exosomes mediate intercellular communication between macrophages and fibroblasts plays an important role in BLM-induced pulmonary fibrosis.

Clusterin as a serum biomarker candidate contributes to the lung fibroblasts activation in chronic obstructive pulmonary disease

Background:Fibrosis in the peripheral airways contributes to airflow limitation in patients with chronic obstructive pulmonary disease(COPD).However,the key proteins involved in its development are still poorly understood.Thus,we aimed to identify the differentially expressed proteins(DEPs)between smoker patients with and without COPD and elucidate the molecular mechanisms involved by investigating the effects of the identified biomarker candidate on lung fibroblasts.Methods:The potential DEPs were identified by isobaric tags for relative and absolute quantitation(iTRAQ)-based proteomic analysis.The messenger RNA and protein levels of clusterin(CLU)in COPD patients and 12%cigarette smoke extract(CSE)-treated human bronchial epithelial cells were determined at the indicated time points.Furthermore,anin vitro COPD model was established via the administration of 8%CSE to normal human lung fibroblasts(NHLFs)at indicated time points.The effects of CSE treatment andCLU silencing on proliferation and activation of lung fibroblasts were analyzed.Results:A total of 144 DEPs were identified between COPD patients and normal smokers.The iTRAQ-based proteomics and bioinformatics analyses identified CLU as a serum biomarker candidate.We also discovered that CLU levels were significantly increased(P<0.0001)in Global Initiative for Obstructive Lung Disease II,III,and IV patients and correlated(P<0.0001)with forced expiratory volume in 1 s(R=-0.7705),residual volume(RV)(R=0.6281),RV/total lung capacity(R=0.5454),and computerized tomography emphysema(R=0.7878).Similarly,CLU levels were significantly increased in CSE-treated cells at indicated time points(P<0.0001).The CSE treatment significantly inhibited the proliferation,promoted the inflammatory response,differentiation of NHLFs,and collagen matrix deposition,and induced the apoptosis of NHLFs;however,these effects were partially reversed byCLU silencing.Conclusion:Our findings suggest that CLU may play significant roles during airway fibrosis in COPD by regulating lung fibroblast activation.

The role of calcineurin in the lung fibroblasts proliferation and collagen synthesis induced by basic fibroblast growth factor

Objective To investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).Methods We used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using 32P-labelled substrate. In the primary culture of lung fibroblasts, 3H-thymidine (3H-TdR) and 3H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DMA and collagen synthesis stimulated by bFGF.Results We found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1±2.0 pmol Pi/mg pr/min). CsA (10^(-8)-10^(-6) mol/L) inhibited lung fibroblast 3H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20% , 46% and 66% (P< 0.01). CsA (10^(-7) -10^(-6) mol/L) inhibited 3H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37% (P<0. 01). In a culture medium, CsA (10^(-8)-10^(-6) mol/L) inhibited 3H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%, 29% (P<0. 05) and 56% (P<0. 01). CsA (10^(-7) mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts (P<0. 01).Conclusions Calcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.

Cigarette induced release of exo-miR-34a from 16HBE vesicles targeting CASP2 promoted the proliferation of COPD MRC-5 cell

Objective:To explore how cigarette smoke extract(CES)regulates the expression of exosomal miR-34a in 16 HBE bronchial epithelial cells,thus affecting the proliferation of MRC-5 lung fibroblasts.Methods:CES was prepared from commercially available cigarettes,and 16HBE cells were treated with CES.The exosomal miR-34a collected from Yipeng Ding,Chief Physician,M.D..the supernatant was used for MRC-5 cell culture.The expression level of exosomal miR-34a was detected by RT-PCR.The proliferation ability of MRC-5 cells was determined by CCK-8 cell counting kit.The expression of CASP2 was detected by Western blot,and the target binding of miR-34a and CASP2 gene was verified by dual luciferase.Results:Under the transmission electron microscope,the exosomes in the supernatant of 16 HBE were spherical,with a particle size of about 100 nm;after CES treatment,the expression of exosomal miR-34a significantly increased.Further research showed that the exosomal miR-34a induced by CES can promote the proliferation of MRC-5 cells;miR-34a and CASP2 have a target binding relationship;miR-34a mimic significantly inhibited the expression of CASP2.Conclusion:In CES-induced 16HBE cells,exosomal miR-34a plays a key role in fibroblast proliferation through target binding with the CASP2 gene.

EFFECTS OF CALCIUM AND CALMODULIN INHIBITORS ON THE ABNORMAL PROLIFERATION OF LUNG FIBROBLAST

The effects of alveolar macrophage (Am) conditioned media from interstitial lung disease (ILD) patients on fibroblast (FB), and the role of calcium (Ca2+) blockers and calmodulin (CaM) inhibitors on the proliferation of lung FB were studied. We found that the AM conditioned media could stimulate FB cell proliferation and this effect could be abolished by Ca2+ blockers and CaM inhibitors. The results indicated that AM was in activated state in ILD and released some kinds of cytokines to stimulate the proliferation of FB, and Ca,2+ CaM were partially responsible for these actions.

Genomic,Lipidomic,and Metabolomic Analysis of Cyclooxygenase-null Cells:Eicosanoid Storm,Cross Talk,and Compensation by COX-1

The constitutively-expressed cyclooxygenase 1（COX-1） and the inducible COX-2 are both involved in the conversion of arachidonic acid（AA） to prostaglandins（PGs）.However,the functional roles of COX-1 at the cellular level remain unclear.We hypothesized that by comparing differential gene expression and eicosanoid metabolism in lung fibroblasts from wild-type（WT） mice and COX-2^（-/-） or COX-1^（-/-） mice may help address the functional roles of COX-1 in inflammation and other cellular functions.Compared to WT,the number of specifically-induced transcripts were altered descendingly as follows：COX-2^（-/-） 〉 COX-1^（-/-） 〉 WT ＋ IL-1β.COX-1^（-/-） or COX-2^（-/-） cells shared about 50%of the induced transcripts with WT cells treated with IL-1β,respectively.An interactive ＂anti-inflammatory,proinflammatory,and redox-activated＂ signature in the protein-protein interactome map was observed in COX-2^（-/-） cells.The augmented COX-1mRNA（in COX-2^（-/-） cells） was associated with the upregulation of mRNAs for glutathione S-transferase（GST）,superoxide dismutase（SOD）,NAD（P）H dehydrogenase quinone 1（NQO1）,aryl hydrocarbon receptor（AhR）,peroxiredoxin,phospholipase,prostacyclin synthase,and prostaglandin E synthase,resulting in a significant increase in the levels of PGE2,PGD2,leukotriene B4（LTB4）,PGF（1α）,thromboxane B2（TXB2）,and PGF（2α）.The COX-1 plays a dominant role in shifting AA toward the LTB4 pathway and anti-inflammatory activities.Compared to WT,the upregulated COX-1 mRNA in COX-2^（-/-） cells generated an ＂eicosanoid storm＂.The genomic characteristics of COX-2^（-/-） is similar to that of proinflammatory cells as observed in IL-1β induced WT cells.COX-1^（-/-） and COX-2^（-/-） cells exhibited compensation of various eicosanoids at the genomic and metabolic levels.