The Research Of Age-related Pulmonary Emphyse Mice Lacking Hydrolase Domain Containing 2 Gene And The Occurring Metabolism

The research of age-related pulmonary emphyse mice lackingα/βphydrolase domain containing 2 gene and the occurring metabolismIntroductionPulmonary emphysema is a major subset of the clinical entity chronic obstructive pulmonary disease and is characterized by destruction of alveolar architecture,loss of lung elasticity,and enlargement of alveolar airspaces.Pulmonary emphysema is a major cause of morbidity and death worldwide.Many of the molecular insights into the pathogenesis of pulmonary emphysema come from the study of rodent models,including genetically manipulated mice.Several lines of research are relevant to the study of emphysema.First,developmental defects in lung morphogenesis lead to lung emphysema.For example,a deficiency of platelet-derived growth factor A chain in mice resulted in loss of alveolar myofibroblasts,leading to failure of alveogenesis.Lung emphysema developed in these mice secondary to the failure of alveolar septation,characterized by lack of elastin fibers and alveolar septa.However,this developmentally acquired emphysema is fundamentally different from the destruction of normal,mature fibers seen in acquired emphysema.Second,damage can be attributed to an imbalance in the endogenous protease/antiprotease equilibrium,promoting tissue hydrolysis that leads to alveolar septal rupture by digestion of septal matrix proteins.The evidence that excessive proteases in the lung cause emphysema is strong and continues to accumulate as new animal models of emphysema are identified.From the initial observations that a papain aerosol caused emphysema,to more recent models based on genetic manipulation, excessive protease activity or deficient antiprotease activity causes emphysema.Third, the oxidant/antioxidant imbalance,an excess of oxidants and free radicals,in the lung promotes cellular and tissue damage and is the major initiator of the disease process. When the balance between oxidants and antioxidants shifts in favor of the former,from either an excess of oxidants and/or depletion of antioxidants,oxidative stress occurs. Oxidative stress produces not only direct injurious effects in the lungs but also activates molecular mechanisms that initiate lung inflammation.Fourthly,it has recently been recognized that alveolar cell apoptosis is a crucial step in emphysgma.Increased levels of structural cell apoptosis in lungs were also demonstrated in animal models of emphysema.For example,Kasahara et al.demonstrated that VEGF receptor signaling is required for maintenance of the alveolar structures,and that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema..Furthermore, novel concepts pertaining to senescence and aging or autoimmunity are currently of growing interest.For example,deficiency of the senescence marker protein-30 (SMP-30) resulted in emphysema.Evidence for acquired immunity in animal models was also demonstrated by the importance of the T cell inflammatory response in the development of smoke-induced emphysema,although some of the literature is contradictory(for a review see).Finally,inflammatory responses can cause protease/antiprotease imbalance,oxidase antioxidase imbalance,and increased apoptosis,leading to emphysema in mouse models.These different pathogenetic mechanisms can operate individually or in concert to increase alveolar destruction or compromise maintenance and repair of the alveolar structure.In mice,three cDNAs encoding proteins containing anα/βphydrolase fold were cloned from an emphysematous mouse lung cDNA library;they were named as lungα/βhydrolase(Labh) 1,2,and 3.These are now termed asα/βhydrolase domain containing(Abhd) 1,2 or 3.All three Abhd proteins are shown to have a single predicted amino- terminal transmembrane domain.Although proteins in this family generally act as enzymes(such as lipases),the specific functions of the three Abhd proteins are unknown.We previously established an Abhd2 deficient mouse line by gene trapinsertional mutagenesis.Although the Abhd2 protein was suggested to be expressed in the alveolar typeâ…¡cells,its function in the lung remains to be elucidated. In this study,we demonstrated that the Abhd2 deficient mice developed spontaneous, gradual emphysema in the lung.Materals and methods1.Gene trap miceThe gene trap mouse line,Abhd2^Gt/Gt,in which the mouse Abhd2 gene was disrupted,was established as described previously.In this study,we used mice of the F10 generation.PCR analyses for genotyping of miceGenotyping was performed by PCR using tail genomic DNA as a template according to the method described previously.,.?,^...;...,.,.?.,,2.RNA analysismRNA levels were assessed using Northern blotting and RT-PCR.Northern blot analysis was performed as described previously.In the RT-PCR assays,RNA samples were reverse transcribed according to the manufacturer's instructions for ThermoScript^TM RT-PCR System(Invitrogen^TM),and gene-specific primers (Supplemental Table 1) were used to amplify selected regions of each target moiety. For each gene,the optimal numbers of cycles that will produce a quantity of product that is directly proportional to the quantity of input mRNA was determined experimentally.Hprt was used as an internal standard.Amplified PCR products were detected using ethidium bromide gel electrophoresis and the intensity of the bands was quantified by densitometry using ImageJ software(version 1.38,a program inspired by NIH image;http://rsb.info.nih.gov/ij/docs/index.html).3.Western blot analysisThe lung was homogenized in lysate buffer[HEPES 50 mmol/l,pH 7.4,NaCl 150 mmol/l,Triton X-100 0.1%,glycerol 10%,NaF 1 mmol/l,sodium orthovanadate 2 mmol/l,EDTA 1 mmol/l,and a protease inhibitor cocktail(1:100 dilution); Sigma-Aldrich,St.Louis,MO,USA].The extracts(60μg protein per lane) were applied to 12%SDS-PAGE and transferred to an Immobilon polyvinylidene difluoride filter(Millipore,Billerica,MA,USA).Primary antibodies were used at the indicated dilutions:goat anti-mouse Sftpal antibody(diluted 1:500;Santa Cruz Biotechnology, Santa Cruz,CA,US),goat anti-mouse Sftpb antibody(diluted 1:500;Santa Cruz Biotechnology,Santa Cruz,CA,US),rabbit anti-mouse Sftpc antibody(diluted 1:200; Santa Cruz Biotechnology,Santa Cruz,CA,US),and mouse anti-mouse Sftpd antibody (diluted 1:500;Santa Cruz Biotechnology,Santa Cruz,CA,US).HRP-conjugated secondary antibody(diluted 1:200;Vector MOM Kit(Vector Laboratories, Burlingame,CA,USA) was used for detection.The intensity of the bands was quantified by densitometry using Image J software.4.Histological and morphometrical analysisThe lungs of terminally anesthetized mice were removed and inflated with 4% paraformaldehyde in PBS to a pressure of 25 cm H₂O.The tissues were then embedded in paraffin and 2 m-thick sections were stained with hematoxylin and eosin.Air space enlargement was quantified by the mean linear intercept(MLI) in 20 randomly selected fields of tissue sections.Measurements were performed on mice at 2 months,4 months,6 months,and 12 months of age.5.Immunohistochemistry and immunofluorescent analysisMice were killed after anesthesia with ether.Their lungs were excised,fixed in 4% paraformaldehyde in PBS,and embedded in paraffin for immunohistochemical and immunofluorescent analyses.Immunohistochemistry was performed using mouse anti-P180 lamellar body protein antibody(3C9) for alveolar typeâ…¡cells(diluted1:250; GeneTex,Inc.San Antonio,TX,USA),rat anti-mouse F4/80 antibody for macrophages (eBioscience,San Diego,CA,USA),and rabbit anti-beta Galactosidase antibody (diluted 1:200;Abcam,Cambridge,UK).Primary antibody was detected with HRP-conjugated anti-mouse secondary antibody(diluted 1:200;Vector MOM Kit, Vector Laboratories,Burlingame,CA,USA),peroxidase conjugated anti-rat secondary antibody(Histofine Simple stain mouse MAX-PO(Rat),Nichirei corporation,Tokyo, Japan),and biotinylated anti-rabbit secondary antibody(diluted 1:200;Vectastain ABC Kit,Vector Laboratories,Burlingame,CA,USA),according to the manufacturer's instructions.Immunohistochemistry for elastin was carried out using Elastica van Gieson staining kit(Merck KGaA,64271 Darmstadt,Germany).To determine whether alveolar typeâ…¡cells expresβ-galactosidase,immunofluorescent analysis was carried out using mouse monoclonal anti-p180 lamellar body protein or rabbit anti-beta galactosidase antibody.For visualization,the following secondary antibodies,anti-mouse Alexa Fluor594 or anti-rabbit Alexa Fluor488-conjugated antibodies(Molecular Probes,Eugene,OR),were used.The nuclei were stained with 4'-6-diamidino-2'-phenylindole(DAPI)(0.1μg/mL;Sigma-Aldrich,Tokyo,Japan). Stained cells were observed using a BZ-9000 microscope(KEYENCE,Osaka,Japan). We counted macrophages and alveolar typeâ…¡cells that were identified by anti-mouse F4/80 and anti-180 immunostaining,respectively.Cell counts were determined by counting 10 random fields each at a magnification of x4006.Bronchoalveolar lavage(BAL) fluid and Lung homogenate Mice were deeply anesthetized with intraperitoneal pentobarbital sodium(100 mg/kg),and the distal aorta was cut to exsanguinate each animal.The chest of the animal was opened,and bronchoalveolar lavage(BAL) was performed by intratracheal injection of sterile saline.Lungs were lavaged three times with 1 ml saline,and recovered fluid from each sample was immediately centrifuged(450 x g for 10 min, 4℃) to remove cells.The supernatant was used to determine phosphatidylcholine(PC), disaturated phosphatidylcholine(DSPC) and protein concentrations.After BAL,the lungs were homogenized in saline and subjected to the above determinations.7.Biochemical measurementsPC content in BAL fluids and lung homogenates was directly determined with a Phospholipids-C assay kit(Wako Pure Chemical Industries,Ltd.,Osaka,Japan).DSPC was isolated from lipid extracts of BAL fluids and lung homogenates.Lipids were reacted with osmium tetroxide,followed by PC determination.Total protein in BAL fluids and lung homogenates was measured by the method of Smith et al..8.TUNEL assayTo detect apoptosis,the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling(TUNEL) assay was performed using an ApopTag Peroxidase in situ apoptosis detection kit(S7100;Chemicon International, Temecula,California,USA).9.Statistical AnalysisAll values are given as means±SE.Differences between groups were tested by Student's t tests.P values＜0.05 were considered to be statistically significant.Results1.Expression ofβ-gal in the alveolar typeâ…¡cellIn the Abhd2^Gt/Gt,а/β-geo gene in the trap vector was inserted into the mouse Abhd2 locus.Thus,we can determine whether the Abhd2 gene is expressed actually in the alveolar typeâ…¡cells in the lung by immunofluorescence analysis using the anti-beta galactosidase antibody and the anti-P180 antibody.X-gal positive cells were co-stained with anti-P180 antibody suggesting that the alveolar typeâ…¡cells express Abhd2 (Supplemental Figure1A).2.Morphology and histochemical Analysis Histological assessment of lungs from Abhd2^Gt/Gt mice at the light microscopic level demonstrated no detectable abnormalities in lung morphology before 6 months of age.Emphysematous changes in lungs were observed in Abhd2^Gt/Gt mice at 6 and12 months of age(Fig.1A).The airspace enlargement in the tissue sections was morphometrically quantified at 2,4,6 and 12 months of age.The mean values of the mean linear intercept in Abhd2^Gt/Gt mice were slightly greater than those in wild-type mice at 6 and 12 months of age.The number of alveolar typeâ…¡cells in the lung of Abhd2^Gt/Gt mice was lower than that of control mice at 12 months of age,but not at 6 months of age.When the sections were stained by EVG stain for elastin fibers,we observed a decrease in the amount of elastin fibers.Although pulmonary surfactant is essential for normal lung mechanics and gas exchange in the lung,the overall general effect of surfactant phospholipids appears to be more complex,including anti-inflammatory effects.Thus we analyzed the number of macrophages in the lung of Abhd2^Gt/Gt mice.Numbers of macrophages were unchanged up to 4 months of age,but accumulation of macrophages was observed at 6 and 12 months of age.Thus Abhd2^Gt/Gt mice developed spontaneous gradual emphysema,which is akin to the pace of emphysema that develops in humans.3.Lipids and proteins in lung and BAL from Abhd2^Gt/Gt miceTo determine whether the Abhd2 deficiency disrupts surfactant phospholipids synthesis and secretion,the amounts of PC,DSPC and protein in BAL fluids and lung homogenates were assessed.In BAL fluid,the concentrations of PC in Abhd2^Gt/Gt mice at 2,6,and 12 months of age were lower than those in the wild-type,whereas the amounts of DSPC and protein were approximately normal in Abhd2^Gt/Gt mice.In lung tissues,there was no significant difference in amounts of PC,DSPC or protein between the wild-type and Abhd2 mice,except for a lower amount of PC in Abhd2 mice at 6 months of age.These results suggest that PC secretion into BAL was slightly disturbed in the lungs of Abhd2^Gt/Gt mice,and that the low concentration of PC is due to the decrease of phospholipids other than DSPC.We also analyzed the expression levels of surfactant proteins(SPs).There were no significant differences in the mRNA and protein expression of SP-A,SP-B,SP-C,or SP-D in Abhd2^Gt./Gt mouse lungs compared with wild-type lungs.4.Protease and anti-protease alteration To define the mechanisms of emphysema,we first compared the expression of key MMPs,cathepsins,and TIMPs in wild-type and Abhd2^Gt/Gt mice using semi-quantitative RT-PCR.analysis.As shown in Fig.2A,the expression levels of MMP-9,MMP-12,MMP-13,MMP-14,cathepsin B,cathepsin K,and cathepsin S were statistically significantly increased in the lungs of Abhd2 ^Gt/Gt mice at 12 months of age. On the other hand,the level of TIMP3 was decreased in the lungs of Abhd2^Gt/Gt mice. The expression levels of MMP-2,cathepsin H,cathepsin L,TIMP-1,TIMP-2,TIMP-4 were unchanged between the wild-type and Abhd2^Gt/Gt mice.These data suggest that a protease/antiprotease imbalance is responsible for emphysema in the lungs of Abhd2^Gt/Gt mice.5.Oxidant and anti-oxidant alterationWe then determined the mRNA levels of 1-antitrypsin,Nrf2,Nox3,SOD and TLR4.However,the expression levels of these were unchanged in the lungs of Abhd2^Gt/Gt mice(data not shown),suggesting that oxidant/antioxidant imbalance is not the cause of emphysema in Abhd2^Gt/Gt mice.6.Inflammatory cytokine alterationIncreased lung inflammation and the overexpression of soluble mediators are important in the pathogenesis of human,as well as experimental,emphysema. Therefore,we investigated whether mice exhibited increased levels of cytokines.As expected,Abhd2^Gt/Gt mouse lungs exhibited increased IL-1β,IL-6,IL-13,and TNF-compared with wild-type lungs.7.Apoptosis in the lungsIncreased levels of structural cell apoptosis in lungs have also been demonstrated in animal models of emphysema.Thus we examined the expression of INF-γ, TGF-β,integrinβ6,caspase 3 and TNF-α.As shown in Fig.2B,the expression levels of INF-αand TGF-βwere increased,while the level o VEGF was decreased in the lungs of Abhd2^Gt/Gt mice.We then analyzed the number of apoptotic cells.The numbers of TUNEL stain positive cells in Abhd2^Gt/Gt mice were remarkably increased compared with those in the wild-type at 6 and 12 months of age.These results suggest that apoptosis is responsible for emphysema in the lungs of Abhd2^Gt/Gt mice. Conclusions1.Abhd2 was expressed in the alveolar typeâ…¡cells.Abhd2 deficiency resulted in a decreased level of phosphatidylcholine in the bronchoalveolar lavage.2.These mice developed spontaneous gradual progression of emphysema,due to increased macrophage infiltration,increased inflammatory cytokines,a protease/antiprotease imbalance and enhanced apoptosis.This phenotype is more akin to the pace of emphysema that develops in humans.3.derangement in alveolar phospholipid metabolism can induce emphysema,and that Abhd2 plays a critical role in maintaining lung structural integrity.