Enhanced Deposition of Low-Molecular-

Enhanced Deposition of Low-Molecular-Weight Hyaluronan in Lungs of Cigarette Smoke–Exposed MiceKen R.Bracke1*,Mieke A.Dentener2*,Eleni Papakonstantinou3,Juanita H.J.Vernooy2,Tine Demoor1,Nele S.Pauwels1,Jack Cleutjens4,Robert Jan van Suylen4,Guy F.Joos1,Guy G.Brusselle1,and Emiel F.M.Wouters21Laboratory for Translational Research in Obstructive Pulmonary Diseases,Department of Respiratory Medicine,Ghent University Hospital,Ghent, Belgium;2Nutrition and Toxicology Research Institute Maastricht(NUTRIM),Department of Respiratory Medicine,and4Department of Pathology, University Hospital Maastricht,Maastricht,The Netherlands;and3Department of Pharmacology,School of Medicine,Aristotle University of Thessaloniki,GreeceChronic obstructive pulmonary disease(COPD)is characterized by infiltration of inflammatory cells,destruction of lung parenchyma, and airway wall remodeling.Hyaluronan(HA)is a component of the extracellular matrix,and low-molecular-weight(LMW)HA frag-ments have proinflammatory capacities.We evaluated the presence of HA in alveolar and airway walls of C57BL/6mice that were exposed to air or cigarette smoke(CS)for4weeks(subacute)or24weeks (chronic).We measured deposition of the extracellular matrix pro-teins collagen andfibronectin in airway walls and determined the molecular weight of HA purified from lung tissue.In addition,we studied the expression of HA-modulating genes by RT-PCR.HA staining in alveolar walls was significantly enhanced upon chronic CS exposure,whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure.This differed from the deposi-tion of collagen andfibronectin,which are only elevated at the chronic time point.In lungs of CS-exposed mice,the molecular weight of HA clearly shifted toward more LMW HA fragments.CS exposure significantly increased the mRNA expression of the HA synthase gene Has3in total lung tissue,whereas the expression of Has1was decreased.These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW)HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes.This may contribute to airway wall remodeling and pulmonary inflammation in COPD. Keywords:hyaluronan;COPD;cigarette smoke;inflammation;airway wall remodelingChronic obstructive pulmonary disease(COPD)is a disease of the airways and lungs that is characterized by a progressive airflow limitation that is not fully reversible(1).The airflow limitation is associated with an abnormal inflammatory re-sponse of the lungs to noxious particles and gases.Significant extrapulmonary effects also contribute to the severity of the disease().The prevalence of COPD is in-creasing,and COPD will soon be the third leading cause of death worldwide(2,3).The pathology of COPD includes obstruction of the small airways(bronchiolitis)and destruction of the lung parenchyma(emphysema).Patients with COPD also show pathologically distinct structural alterations of the small airways (airway wall remodeling)(4).The molecular and cellular mech-anisms leading to cigarette smoke(CS)-induced inflammation, pulmonary emphysema,and airway wall remodeling have not been elucidated.Cigarette smoking is by far the most important risk factor for the development of COPD,and it has been shown that chronic exposure to CS leads to lung inflammation with in-creased numbers of inflammatory cells,including macrophages (5,6),dendritic cells(DCs)(7–9),neutrophils(10,11),and CD81 T lymphocytes(12).Pulmonary emphysema and airway wall remodeling in COPD are associated with damage and ineffective repair of the extracellular matrix(ECM).Hyaluronan(HA)is a compo-nent of the ECM that is present at enhanced levels in sputum of patients with COPD(13).HA exists in healthy tissues as a high-molecular-weight(HMW)nonsulfated glycosaminoglycan poly-mer,composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine.In the ECM,HA plays a role in water homeostasis,plasma protein distribution,and matrix structure(14).HA has additional biological functions that are reported to be dependent on its molecular size.Oligosaccha-rides of HA can induce angiogenesis and endothelial cell proliferation(15)and can activate DCs(16).Low-molecular-weight(LMW)HA fragments,with an average molecular mass of250kD,exhibit proinflammatory capacities by stimulating production of cytokines,chemokines,and matrix metallopro-teinase–12by macrophages(17,18).In contrast,HMW HA has protective effects for osteoarthritis and rheumatoid arthritis (19).Under normal conditions,there is a continuous turnover of HA.Three isoforms of human HA synthases have been identified,all differing in their enzymatic properties and the length of the HA chain that is formed(20).HA synthase(Has)1 and Has2synthesize HMW HA,whereas Has3creates LMW HA fragments.HA is broken down by hyaluronidases,of which hyaluronidase(Hyal)1and Hyal2are the most important in somatic tissues.Hyal2degrades HMW HA to LMW HA fragments,whereas Hyal1cleaves LMW HA to oligosaccha-rides(21).In this study,we report the effects of subacute and chronic CS exposure on alveolar and bronchial deposition of the glycosaminoglycan HA in a murine model of COPD.We measured the bronchial deposition of the ECM proteins colla-gen andfibronectin and compared the time courses of the CS-induced modulation of deposition of HA versus ECM proteins. We determined the molecular weight of hyaluronan in lungs of air-and CS-exposed mice.In addition,we analyzed the in vivo effect of CS on mRNA expression of HA synthases and hyaluronidases in total lung tissue and isolated lung macro-phages.(Received in original form November3,2008and infinal form July15,2009) This work was supported by an unrestricted grant from GSK,the Fund for Scientific Research in Flanders(FWO Vlaanderen,Research Project G.0011.03), the Strategic Basic Research(SBO-IWT/020203)and the Concerted Research Action of the University of Ghent(BOF/GOA01251504and01G01009).K.R.B.is a post-doctoral researcher of the Fund for Scientific Research in Flanders(FWO Vlaanderen).*These authors contributed equally to this work.Correspondence and requests for reprints should be addressed to Ken Bracke, Department of Respiratory Medicine,Ghent University Hospital,7K12IE De Pintelaan,185B-9000Ghent,Belgium.E-mail:ken.bracke@UGent.beAm J Respir Cell Mol Biol Vol42.pp753–761,2010Originally Published in Press as DOI:10.1165/rcmb.2008-0424OC on August12,2009 Internet address:MATERIALS AND METHODSAnimalsMale C57BL/6mice,6to8weeks old,were purchased from Harlan (Zeist,The Netherlands).The local Ethics Committee for animal experimentation of the faculty of Medicine and Health Sciences (Ghent,Belgium)approved all in vivo manipulations.Smoke ExposureMice(n58per group)were exposed whole body to CS as described previously(22–24).Briefly,groups of eight mice were exposed to the tobacco smoke offive cigarettes(Reference Cigarette2R4F without filter;University of Kentucky,Lexington,KY)four times a day with 30-minute smoke-free intervals5days per week for4weeks(subacute exposure)or24weeks(chronic exposure).An optimal smoke/air ratio of1:6was obtained.The control groups were exposed to air.Carbox-yhemoglobin in serum of CS-exposed mice reached a nontoxic level of 8.361.4%,compared with1.060.2%in air-exposed mice(n57for both groups),which is similar to carboxyhemoglobin blood concentra-tions of human smokers(25).Bronchoalveolar LavageTwenty-four hours after the last smoke exposure,mice were weighed and killed with an overdose of pentobarbital(Sanofi,Libourne, France),and a tracheal cannula was inserted.Three fractions of 300m l followed by three fractions of1ml of Hanks’balanced salt solution,free of ionized calcium and magnesium but supplemented with0.05mM sodium EDTA,were instilled via the tracheal cannula and recovered by gentle manual aspiration.The six lavage fractions were pooled and centrifuged,and the cell pellet was washed twice and resuspended in1ml of Hanks’balanced salt solution.A total cell count was performed in a Bu¨rcker chamber,and the differential cell counts (on at least400cells)were performed on cytocentrifuged preparations using standard morphologic criteria after May-Gru¨nwald-Giemsa staining.Flow cytometric analysis of bronchoalveolar lavage(BAL) cells was performed to enumerate DCs and CD41and CD81T cells. Labeling of BAL Cells for Flow CytometryCells were preincubated with Fc-receptor blocking antibody(anti-CD16/ CD32,clone2.4G2)to reduce nonspecific binding.Monoclonal anti-bodies used to identify mouse DC populations were allophycocyanine (APC)-conjugated anti-CD11c(N418)and phycoerythrin-conjugated anti–I-A b(AF6-120.1).We discriminated between macrophages and DCs using the methodology described by Vermaelen and colleagues (26).After gating on the CD11c-bright population,two peaks of autofluorescence can be distinguished.Macrophages are identified as the CD11c-bright,highly autofluorescent population and do not express MHCII.DCs are identified as CD11c-bright,low autofluorescent cells, which strongly express MHCII.DCs enumerated by these criteria correspond with myeloid DCs.The following antibodies were used to stain mouse T-cell subpopulations:fluorescein isothiocyanate–conjugated anti-CD4(L3T4),phycoerythrin-conjugated anti-CD8(Ly-2),and APC-conjugated anti-CD3(145-2C11)monoclonal antibodies.All monoclonal antibodies were obtained from BD Pharmingen(San Diego,CA).Cells were incubated with7-amino-actinomycin(7-AAD or viap-robe;BD Pharmingen)for dead cell exclusion.All labeling reactions were performed on ice in FACS-EDTA buffer.Flow cytometry data acquisition was performed on a dual-laser FACS VantageTMflow cytometer running CELLQuest software(Becton Dickinson,Mountain View,CA).FlowJo software() was used for data analysis.Purification and Isolation of Lung MacrophagesTo obtain lung tissue,right heart catheterization and perfusion with saline-EDTA was performed to remove the pulmonary intravascular pool of cells.The right lung was clamped,removed,and collected in ice-cold tissue culture medium for preparation of single-cell suspen-sions.Lung macrophages were isolated as described previously(27).To obtain sufficient amounts of macrophages for RNA preparation,the single-cell suspensions of eight mice were pooled.Pulmonary single-cell suspensions werefirst incubated with Fc-receptor-block,followed by anti-CD11c microbeads(Miltenyi Biotec,Bergisch Gladbach,Ger-many).CD11c1lung cells were enriched after one passage through a VarioMACS magnetic cell separator according to the manufacturer’s instructions.The CD11c2fraction was kept on ice until RNA extraction. The CD11c1fraction was labeled with APC-conjugated anti-CD11c.On the FACS Vantage SE,the CD11c1/highly autofluorescent cells,repre-senting the macrophage population,were separated byfluorescence-activated cell sorting with a70-nm nozzle.HistologyThe left lung wasfixated by infusion of4%paraformaldehyde through the tracheal cannula(22–24).After excision,the lung was immersed in freshfixative for2hours.The lung lobe was embedded in paraffin,cut into3-m m transverse sections,and stained for histologic analysis.Histochemical Localization of HyaluronanHistolocalization of HA was determined on paraffin sections using biotin-labeled HA-binding protein(HABP-b)(Seikagaku,Tokyo, Japan).Sections were subjected to deparaffinization followed by re-hydration.Sections were stained with HABP-b(2m g/ml)at room temperature for1hour.After washing,the DAKO Cytomation Streptavidine ABComplex/HRP system was used according to the manufacturer’s instructions(DAKO,Glostrup,Denmark).Enzymatic reactivity was visualized with the Vector NovaRED peroxidase sub-strate kit(Vector,Burlingame,CA).Sections were lightly counter-stained with hematoxylin and mounted in Faramount(DAKO).No significant staining was detected in sections pretreated with50U/ml Streptomyces hyaluronidase(Calbiochem,San Diego,CA)at378C for 2hours,indicating that this HABP staining reaction was specific for HA.The HABP-b staining in the alveolar walls was semiquantitatively scored by three independent observers(J.C.,R.V.S.,and M.D.)who were unaware of the treatment of the animals.The intensity of the HA staining was expressed in arbitrary units and scored on afive-point scale:05no or hardly any staining,15weak staining at some spots on the alveolar walls,25moderate staining at several spots,35intense staining of most parts of the alveolar walls,and45very intense staining throughout the alveolar walls.The HABP-b staining in the airway walls was scored quantitatively, and three lung sections per animal were examined.The following morphometric parameters(28)were marked manually on the digital representation of the airway:the length of the basement membrane (Pbm),the area defined by the basement membrane(Abm),and the area defined by the total adventitial perimeter(Ao).The total bronchial wall area(WAt)was calculated(WAt5Ao–Abm)and normalized to the squared length of the basement membrane.For the quantification of HA deposition,the area in the airway wall covered by the stain was determined by the software(KS400;Zeiss,Oberkochen, Germany),and its value was calculated as described previously(29). The area of HA was normalized to Pbm.All airways with a Pbm smaller than2,000m m and cut in reasonable cross-sections(defined by a ratio of minimal to maximal internal diameter.0.5)were included. Collagen and Fibronectin MeasurementCollagen in the airway wall was stained using Sirius Red,and the amount offibronectin was determined with a goat antiratfibronectin antibody(Calbiochem,BadsSoden,Germany)using the streptavidin-biotin peroxidase method as previously described(24,29).Bronchial collagen andfibronectin staining was scored quantitatively as described previously for HABP-b staining.Quantification of EmphysemaEmphysema is a structural disorder characterized by destruction of the alveolar walls and enlargement of the alveolar spaces.We determined enlargement of alveolar spaces by quantifying the mean linear in-tercept(L m)after24weeks of smoke exposure as described previously (22,30)using image analysis software(Image J1.33).Only sections without cutting artifacts,compression,or hilar structures(airway or blood vessel with a diameter larger than50m m)were used in the analysis.The L m was measured by placing a1003100m m grid over eachfield.The total length of each line of the grid divided by the754AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL422010number of alveolar intercepts gives the average distance between alveolated surfaces,or the L m.Isolation and Purification of Total Glycosaminoglycansfrom Lung TissueLung tissue specimens were homogenized by a Polytron homogenizer (Kinematica,Luzern,Switserland)(5310-s bursts with1-min intervals in ice)in10ml of25mM Tris-HCl(pH7.6)per gram of tissue. Homogenized tissues were delapidated with4volumes of chloroform/ methanol(1:2volumes).Organic solvents were removed by centrifu-gation(3,(2003g,20min,48C),and the pellet was washed with10ml of ethanol,centrifuged as described previously,and dried at408C for 4hours.The dry pellet was weighed,resuspended in1ml of0.1M Tris-HCl buffer(pH8.0)containing1mM CaCl2,and subjected to protein digestion with0.1KU of Pronase(Streptomyces griseus;Calbiochem). The pronase solution was preincubated for30minutes at608C to eliminate glycosidase activity.Digestion was performed for72hours at 608C by adding equal amounts of pronase at24-hour intervals.The sample concentration was then adjusted to150mM NaCl and10mM MgCl2,and DNA digestion was accomplished by adding400KU DNase I(EC3.1.21.1;Calbiochem)and incubating for16hours at 378C.At the end of the incubation period,the CaCl2concentration of the solution was adjusted to1mM,and the reaction was stopped by adding0.1KU of pronase and incubating the mixture at608C for 24hours.The pH was adjusted to10.0to11.0by the addition of10mM NaOH,and samples were subjected to b elimination in the presence of 1M NaBH4for16hours at458C.Samples were then neutralized with 50%(vol/vol)acetic acid.Total glycosaminoglycans were precipitated with the addition of four volumes of ethanol in the presence of0.1 volume of3M CH3COONa and overnight maintenance at248C, recovered with centrifugation(20min;2,0003g),redissolved in double distilled H2O,and stored at48C.Colorimetric determination of uronic acids was performed according to Bitter and Muir(31). Measurement of Relative Amount of HAin Total GlycosaminoglycansThe relative amount of HA was measured in aliquots containing0.075m g of uronic acids by ELISA(Corgenix,Lynch Wood,Peterborough,UK)as previously described(32).Briefly,ELISA plates coated with HA-binding protein were incubated with samples or standards(1h at room temper-ature)in duplicates,washedfive times with washing buffer,incubated with a solution containing horseradish peroxidase–conjugated HA-binding protein(30min at room temperature),washed againfive times, and incubated with100m l of the substrate solution.After30minutes,the reaction was stopped by adding an equal amount of sulfuric acid(0.36N), and the optical density was measured at450nm(630-nm reference).Agarose Gel Electrophoresis for Measurementof Hyaluronan SizeTotal glycosaminoglycans(6m g of uronic acids),isolated and purified from lung tissue specimens,were analyzed on0.9%agarose gels,as previously described(33).Identification of HA was performed by enzymatic treatment of the samples with hyaluronidase(Streptomyces hyalurolyticus;Seikagaku,Tokyo,Japan)for14hours at408C before gel electrophoresis.The molecular mass of HA was determined using commercially available HA of1,000,250,and50kD(AMS Bio-technology,Oxfordshire,UK).Gels were stained with a solution of 0.005%(wt/vol)stains-all dissolved in50%(vol/vol)ethanol overnight under light-protective cover at room temperature.For destaining,gels were transferred in H2O and incubated for3hours at room temper-ature.RNA Isolation and RT-PCRTotal lung RNA and RNA from isolated lung macrophages was extracted with the RNeasy Mini Kit(Qiagen,Hilden,Germany). cDNA was obtained by reverse transcription of RNA using a3:1ratio of hexa/oligodT primers(Abgene,Epsom,UK).The primer pairs used in this study were designed with Primer Express2.0Software(Applied Biosystems,Foster City,CA)and are listed in Table1.Real-time PCR reactions were performed in duplicate using diluted cDNA template (dilution1:25),0.6pmol of each primer(Table1),and IQ SYBR Green Supermix I dye(Bio-Rad,Hercules,CA)in a total volume of20m l.A standard curve derived from the serial dilutions of a mixture of all samples was included on each plate.Quantitation and real-time detection of the PCR products was followed on an iCycler iQ Real-Time PCR system(Bio-Rad)with the following cycling conditions: 3minutes at958C for Platinum Taq activation and40cycles for the melting(15s at958C)and annealing/extension(45s,608C)steps.These conditions generate specific PCR products of the desired length,which were verified by gel electrophoresis on an ethidium bromide–stained 2%agarose gel.Data were processed using the standard curve–based method.Expression of target genes was corrected by a normalization factor that was calculated based on expression of three reference genes (Hprt1,Ppia,and Rpl13a)using the geNorm applet according to the guidelines and theoretical framework previously described(http:// medgen.ugent.be/z jvdesomp/genorm/)(34).Statistical AnalysisReported values are expressed as mean6SEM.Statistical analysis was performed with Sigma Stat software(SPSS11.0Inc.,Chicago,IL)using nonparametric tests(Kruskall-Wallis and Mann-Whitney U).P values under0.05were considered as significant.TABLE1.GenBank ACCESSION NUMBERS AND OLIGONUCLEOTIDE SEQUENCES OF PRIMERS USEDFOR RT-PCRGene Accession No.Sequences ProductHas1NM_008215Forward:59-ACCTCACCAACCGAATGCTT-3989bpReverse:50-GAAGGAAGGAGGAGGGCG-39Has2NM_008216Forward:59-TGAGTACAAAGAGGTTCGTTCAAGTT-3986bpReverse:59-ATTGTCAGGGTGTGTTTGTTTCC-39Has3NM_008217Forward:59-CTACTTTGTAGCTGCCCAGAATACTG-39116bpReverse:59-GAGTACAAAAAACAGCACCGGAAT-39Hyal1NM_008317Forward:59-CTTCTGCCCCTGGAGGAACT-39141bpReverse:59-GTGTGGAATCCATGTATGCTTTAATG-39Hyal2NM_010489Forward:59-CGAGGACTCACGGGACTGA-39150bpReverse:59-GCTGAGTTAGGTAATTCTTGAGGTATTG-39Rpl13a NM-009438Forward:59-CACTCTGGAGGAGAAACGGAAGG-39182bpReverse:59-GCAGGCATGAGGCAAACAGTC-39Ppia NM_008907Forward:59-TTCCTCCTTTCACAGAATTATTCCA-3975bpReverse:59-CCGCCAGTGCCATTATGG-39Hprt1NM_013556Forward:59-AGCTACTGTAATGATCAGTCAACG-39198bpReverse:59-AGAGGTCCTTTTCACCAGCA-39Definition of abbreviations:Hprt15hypoxanthine phosphoribosyltransferase1;Ppia5cyclophilin A/peptidylprolyl isomerase A;Rpl13a5ribosomal protein L13a.Bracke,Dentener,Papakonstantinou,et al.:Hyaluronan in a Murine Smoke Model of COPD755RESULTSCS-Induced Increase of Inflammatory Cells in BAL FluidUpon subacute (4-wk)and chronic (24-wk)exposure to CS,the total cell number in the BAL fluid was significantly increased,compared with air-exposed control animals (Figure 1A).Expo-sure to CS caused a significant accumulation of macrophages,DCs,neutrophils,CD41T lymphocytes,and CD81T lympho-cytes in the BAL fluid (Figures 1B–1F).Increased Hyaluronan Deposition in Alveolar and Airway Walls upon CS ExposureHistochemical localization using HA binding protein revealed little HA staining in alveolar walls of air-exposed mice.Semi-quantitative scoring of the HA staining in alveolar walls showed only slightly enhanced HA deposition upon subacute (4-wk)CS exposure,whereas chronic (24-wk)CS exposure caused a signif-icant increase in the deposition of HA (Figure 2).Histochemical localization of HA revealed constitutive staining in airway walls of air-exposed mice.Quantitative scoring using an image analyzer showed that subacute (4-wk)and chronic (24-wk)exposure to CS caused a significant in-crease in peribronchial deposition of HA compared with air-exposed animals (Figure 3).Increased Bronchial Deposition of Collagen and Fibronectin upon Chronic CS ExposurePeribronchial deposition of protein components of the ECM was studied on Sirius Red–and antifibronectin-stained histo-logic sections to reveal collagen in fibronectin,respectively.Subacute (4-wk)exposure to CS did not induce an increase in the peribronchial deposition of collagen and fibronectin (Figure 4).Upon chronic (24-wk)exposure to CS,there was a significant increase in deposition of collagen and fibronectin in airway walls as compared with air-exposed control mice (Figure 4).Development of Pulmonary Emphysema upon Chronic CS ExposurePulmonary emphysema is characterized by the destruction of alveolar walls due to damage to the lung parenchyma,leading to enlargement of alveolar spaces.Therefore,we quantified em-physematous lesions by measuring the mean linear intercept (L m ).Chronic (24-wk)exposure to CS clearly induced pulmonary emphysema in wild-type animals,as evidenced by a significant increase in the L m (air 38.060.9m m versus CS 41.960.5m m;P ,0.05).Increased LMW Hyaluronan Fragments in Lung Tissue of CS-Exposed MiceTotal glycosaminoglycans were isolated and purified from lung tissue specimens.Measurement of the content of uronic acids in lung samples revealed that they contain 7.060.8m g of uronic acids per mg of dry defatted lung tissue.There were no significant differences in the content of total glycosaminogly-cans in dry defatted lung tissue between the different groups tested (data not shown).The content of HA in total glycosaminoglycans isolated and purified form lung tissue specimens was measured in 0.075m gofFigure 1.Effect of cigarette smoke-exposure on cell differentiation in bron-choalveolar lavage (BAL)fluid.Total BAL cells and cell differentiation in BAL fluid upon subacute (4-wk)and chronic (24-wk)exposure to air or cigarette smoke.(A )Total BAL cells.(B )Macro-phages.(C )Dendritic cells.(D )Neutro-phils.(E )CD41T lymphocytes.(F )CD81T lymphocytes.Results are expressed as means 6SEM;n 58animals per group (*P ,0.05).756AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 422010uronic acids using ELISA.There were no significant differences in the relative content of HA in total glycosaminoglycans in the lung of subacute (4-wk)CS-exposed mice.However,we ob-served a significant increase of the relative content of HA after chronic (24-wk)CS exposure (Figure 5A).We analyzed the molecular mass of HA that was isolated and purified from lung tissue specimens by agarose gel electro-phoresis.HA isolated from the lungs of air-exposed mice migrated as a broad band,with an average molecular mass of 500kD.HA isolated from the lungs of CS-exposed mice exhibited a lower molecular mass of an average of 70kD after subacute (4-wk)and chronic (24-wk)CS exposure (Figure 5B).Expression of HA-Modulating Genes in Total Lung Tissue upon CS ExposureExpression of HA synthase genes and hyaluronidases was studied on total lung tissue by RT-PCR.Subacute (4-wk)and chronic (24-wk)CS exposure significantly increased the mRNA expression of Has3compared with air-exposed mice (Figure 6C).There was no effect of CS exposure on the expression of Has2(Figure 6B),whereas chronic exposure to CS significantly decreased the mRNA expression of Has1compared with air-exposed control mice (Figure 6A).The mRNA expression of Hyal1was not affected by CS exposure (Figure 6D).However,there was a tendency toward decreased mRNA expression of Hyal2upon chronic CS expo-sure compared with air-exposed mice (Figure 6E).Expression of HA-Modulating Genes in Lung Macrophages upon Subacute CS ExposureExpression of HA synthase genes and hyaluronidases was studied by RT-PCR in macrophages isolated from lungsofFigure 2.Effect of cigarette smoke-exposure on hyaluronan (HA)deposition in alveolar walls.(A )Semiquantitative scoring of HA de-position in alveolar walls upon subacute (4-wk)and chronic (24-wk)ex-posure to air or cigarette smoke (CS).Results are expressed as means 6SEM;n 58animals per group (*P ,0.05).(B –E )Photomicrographs of HA deposition in alveolar walls upon sub-acute (4-wk)and chronic (24-wk)air or CS exposure.(B )Air exposure,4wk.(C )CS exposure 4wk.(D )Air exposure,24wk.(E)CS exposure,24wk.Figure 3.Effect of CS exposure on peribronchial HA deposition.(A )Quantitative scoring of peribronchial HA deposition upon sub-acute (4-wk)and chronic (24-wk)exposure to air or CS.Results are expressed as means 6SEM;n 58animals per group (*P ,0.05).Pbm 5length of basement membrane.(B –E )Photomicrographs of peribronchial HA deposition upon subacute (4-wk)and chronic (24-wk)air or CS exposure.(B )Air exposure,4wk.(C )CS exposure,4wk.(D )Air exposure,24wk.(E )CS exposure,24wk.Bracke,Dentener,Papakonstantinou,et al .:Hyaluronan in a Murine Smoke Model of COPD 757air-and CS-exposed mice.Upon subacute CS exposure,there was a strong decrease in Has1mRNA in lung macrophages (Figure 7A),whereas Has2and Has3were only slightly de-creased (Figures 7B–7C).In contrast to the HA-synthase genes,the hyaluronidase Hyal2was strongly increased in macrophages from CS-exposed mice (Figure 7E)compared with macro-phages from air-exposed mice.Hyal1mRNA expression was not affected by CS exposure (Figure7D).Figure 4.Effect of CS exposure on peri-bronchial collagen and fibronectin de-position.(A )Quantitative scoring of peribronchial collagen and (F )fibronec-tin deposition upon subacute (4-wk)and chronic (24-wk)exposure to air or CS.Results are expressed as means 6SEM;n 58animals per group (*P ,0.05).Pbm 5length of basement membrane.(B –J )Photomicrographs of peribronchial collagen and fibronectin deposition upon subacute (4-wk)and chronic (24-wk)air or CS exposure.(B ,G )Air ex-posure,4wk.(C ,H )CS exposure,4wk.(D ,I )Air exposure,24wk.(E ,J )CS exposure 24wk.Figure 5.Effect of CS exposure on the relative amount and molecular mass of HA in the lung.Total glycosaminoglycans were isolated and puri-fied from the lungs upon subacute (4-wk)and chronic (24-wk)exposure to air or CS.(A )The relative amount of HA in aliquots of total glycos-aminoglycans containing 0.075m g of uronic acids was determined by ELISA.Results are expressed as means 6SEM;n 58animals per group (*P ,0.05).(B )Representative results obtained after electrophoresis of total glycosaminoglycans corre-sponding to 6m g of uronic acids on 0.9%agarose gels.HA (1m g)of known molecular mass was used as a marker.Gels were stained with a solution of 0.005%(wt/vol)stains-all dissolved in 50%(vol/vol)ethanol and destained in H 2O.758AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 422010。