Patent Application: US-201414762086-A

Abstract:
microfibrillar - associated protein 4 binding antibodies are provided to prevent or to inhibit the proliferation of vascular smooth muscle cells and neointima formation in blood vessels . furthermore , there are provided antibodies that effectively inhibit remodelling of vessels and prevent progression of arteriosclerosis as well as restenosis of vessels . the provided antibodies block the interaction between mf ap4 and integrin receptors .

Description:
mfap4 cross / inks vsmcs to ecm fibrils and induces cellular migration and proliferation through integrin α v β 3 / β 5 ligation in order to elucidate the presence of mfap4 in human vascular tissue with patological remodeling processes sections from a human vein with intimal hyperplasia were obtained from a patient with lower extremity pad that underwent surgical reconstitution following bypass surgery induced restenosis . the neointima appeared with inhomogenous staining for mfap4 . the most intense mfap4 staining was localized closest to the outer periphery of the vessel . a similar staining pattern was found for both α - smooth muscle actin ( α - sma ) and elastin . mfap4 staining appeared to colocalize with the elastic fibers , whereas the α - sma staining was intracellular ( fig1 ). immunostaning detected dispersed integrin α v β 3 staining throughout the neointimal area with highest intensity in capillary endothelium . few intervening cd45 - positive inflammatory cells were also observed . following the inventors demonstrated a high intense immunohistochemical staining for mfap4 and localization to elastic fibers within the normal human mammary artery ( fig2 a ) and mouse aorta ( fig2 b ). a similar staining pattern was found throughout all investigated peripheral muscularized blood vessels from a human organ multi - block . mfap4 synthesis and secretion from fhaosmc &# 39 ; s were observed using cytoimmunohistochemistry ( fig2 c , 2 d ), western blotting ( fig2 e ) and by enzyme - linked immunosorbent assay ( elisa ) quantification in the cell culture supernatant . moreover , both cellular associated ( fig2 c - e ) and secreted ( fig2 f ) mfap4 was increased several fold in parallel with the known marker of differentiation α - sma during & gt ; 100 hrs shift from a proliferative phenotype and into a contractile phenotype in the culture . pull - down experiments were used to detect direct interaction between recombinant mfap4 ( rmfap4 ) and collagen or elastin in a ca 2 + - dependent manner ( fig2 g ) suggesting that the binding takes place through the conserved s1 - binding site in the fred . to identify relevant mfap4 binding integrins , human placenta membrane proteins were affinity purified on immobilized rmfap4 . proteins , which might correspond to integrins according to molecular weight , were eluted from the column and integrin monomers α v , β 1 , β 3 , and β 5 were detected specifically in the collected fractions ( fig3 a and b ). similar results were obtained using fluorescence - activated cell sorting ( facs ) analysis of fhaosmc ( fig3 c ). a cellular adhesion assay following demonstrated that calcein am fluorogenic dye labeled fhaosmcs attached to rmfap4 , fibronectin , and laminin and failed to adhere to bovine serum albumin ( bsa ) ( fig3 d ). the synthetic peptide grgdsp completely inhibited cellular attachment of to rmfap4 , whereas the control peptide sdgrg showed no significant inhibition ( fig3 e ). anti - integrin α v and anti - integrin α v β 3 antibodies completely blocked the cellular adhesion to rmfap4 , while anti - integrin α v β 5 antibodies showed a small but significant reduction in adhesion . in contrast , anti - integrin β 1 had no effect on the cellular adhesion to rmfap4 ( fig3 f ). the integrins were detectable through all tested cel culture conditions . yet , integrin α v and integrin β 5 were coordinately expressed with mfap4 while integrin β 3 expression was diminished when fhaosmcs differentiated from the proliferating to the contractile phenotype . monoclonal anti - mfap4 antibodies were raised in mfap4 −/− mice because the mouse mfap4 homologue has very high sequence similarity to certain regions within the human protein . elisa based assays demonstrated that produced antibodies with reactivity against mfap4 ; anti - mfap4 hg - hyb 7 - 14 and 7 - 18 antibodies clearly bind double ( aga ) and triple ( aaa ) rgd mutated rmfap4 in the chinese hamster ovary ( cho ) cell culture supernatant . in contrast , the rmfap4 detection signals were reduced for the point - mutated proteins when the hg - hyb 7 - 5 was used as capture antibody suggesting that hg - hyb 7 - 5 binds to an epitope covering the rgd sequence in rmfap4 . hg - hyb 7 - 5 and hg - hyb 7 - 14 both prohibited the cellular adhesion to rmfap4 . this latter observation suggests that hg - hyb 7 - 14 may bind at close proximity to the rgd site . it was further observed that focal adhesions and cellular stress fibers ( vinculin and f - actin , respectively ) formed within 20 hrs of exposure to either fibronectin or rmfap4 , but not with poly - d - lysin . inhibition of focal adhesion and stress fiber formation was observed when fhaosmcs were incubated with the blocking antibodies . cellular migration and proliferation of vsmcs are induced by mfap4 and inhibited by mfap4 blocking antibodies the fhaosmc migration was increased almost 2 - fold towards immobilized rmfap4 ( fig4 a and b ). incubation with hg - hyb 7 - 5 or 7 - 14 antibodies reduced the numbers of migrating cells significantly while no effect was seen when using the non - blocking hg - hyb 7 - 18 antibody or the isotype control ( fig4 a and c ). the effect of rmfap4 on proliferating fhaosmc was further assessed using a thiazoyl blue tetra - zolium bromide ( mtt )- assay . the cells were allowed to proliferate for 48 hrs , either in the presence or absence of 5 ng / ml platelet - derived growth factor - bb ( pdgf - bb ) and the proliferation was significantly induced when cells were seeded onto either rmfap4 or fibronectin ( fig4 d ). microplates coated with rmfap4 were following blocked with anti - mfap4 antibodies before seeded with fhaosmc . hg - hyb 7 - 5 and 7 - 14 both lead to a significant reduction of cellular proliferation to a non - pdgf - bb treated level ( fig4 e ) in parallel with integrin α v β 3 blocking antibodies ( data not shown ). the mfap4 gene comprises 6 exons coding for the globular fred and a short n - terminal sequence with a free cysteinyl - group for disulphide bridging of dimers ( fig5 a ). mfap4 −/− mice were generated on the cj7 background using insertion of a neomycin cassette into the mfap4 gene replacing exon 1 - 3 coding for the rgd containing n - terminal domain and a part of the fred ( fig5 b ). deletion was confirmed by southern blotting analysis . additional genotyping by pcr confirmed the presence of a 300 bp gene - deficient fragment and a 224 bp wild - type fragment present in mfap4 −/− and mfap4 +/+ derived mice ( fig5 c ). moreover , rt - pcr analysis using pulmonary tissue lysate demonstrated the lack of mfap4 transcription in the mfap4 −/− mice ( fig5 d ). homozygous mfap4 −/− mice were viable , bred with normal mendelian frequencies , and appeared indistinguishable from wild - type littermates . serum samples were obtained from mice in the 10 th generation . parallelism was observed between the recombinant mouse mfap4 and the wild - type elisa signal ( fig5 e ). mfap4 was absent from the mfap4 −/− circulation and immunohistochemical analysis demonstrated a clear lack of detectable signals from mfap4 −/− mouse tissue . a role for mfap4 in the assembly of microfibrils is previously suggested ( 34 ) but the elastic laminae in the arteries appeared with smooth organized lamellar sheets suggesting that the integrity of the vessel wall was preserved . the unchallenged mfap4 −/− mice appeared with normal heart rate , normal blood pressure , normal circulating cell numbers , and normal blood lipid levels . the resting mean arterial blood pressure ( map ) obtained using chronic indwelling catheters placed in the femoral artery and vein was stable and averaged 98 . 8 ± 2 . 7 mmhg and the heart rate ( hr ) 664 ± 18 bpm in wild - type animals . in mfap4 −/− mice map averaged 105 . 5 ± 3 . 6 mmhg and the hr 661 ± 13 bpm . phenylephrine caused a significant increase in map ( 149 . 5 ± 5 . 1 mmhg and 153 . 1 ± 4 . 2 mmhg ) and a corresponding decrease in hr ; 442 ± 40 bpm and 428 ± 27 bpm , in mfap4 +/+ and mfap4 −/− mice respectively . there was no significant difference between genotypes either at basal levels or at increased blood pressure . decreased vessel diameter and neointima formation in mfap4 −/− mice after carotid artery ligation is associated with reduced vsmc proliferation and infiltration with cd45 positive cells a ligated carotid artery will undergo initial outward remodeling , followed by vessel shrinkage and neointima formation , resulting in narrowing of the lumen ( 35 ). the remodeling responses 14 days and 28 days were compared after left carotid arterial ligation in mfap4 +/+ and mfap4 −/− littermates of the c57bl / 6n strain in order to examine whether the lack of mfap4 affected the arterial response to ligation . transverse sections were obtained 0 . 5 , 1 . 0 , and 1 . 5 mm proximal to the ligature / bifurcation and at corresponding locations in the contralateral vessel and stained with verhoeff - van gieson elastin staining ( fig6 a ). neointimal growth appeared delayed in the mfap4 −/− mice , with very limited or no formation after 14 days , but with neointimal areas comparable to mfap4 +/+ after 28 days ( fig6 b ). furthermore , the external elastic lamina ( eel ) of the ligated mfap4 −/− vessel was significantly decreased compared to the mfap4 +/+ vessel ( fig6 b ). thus , at day 28 the lumen in mfap4 −/− vessels was significantly decreased . no apparent differences in the vessel diameter or in the lumen diameter were observed in the contralateral control arteries . a corresponding lack of outward arterial remodeling was observed 14 days after ligation with mfap4 deficiency in the balb / c background . neointimal areas appeared decreased in the mfap4 −/− mice , however morphometric analysis was not attempted due to rich neovascularization of the neointimal areas in both mfap4 −/− and mfap4 +/+ balb / c mice . immunostaining of the ligated mfap4 +/+ c57bl / 6n vessels revealed that mfap4 localized to medial ( most intense ) as well as to neointimal cells ( fig7 a ). there was no apparent difference in mfap4 immunohistochemical staining intensity between ligated and unligated vessels . rt - qpcr analyses of mfap4 expression were further performed in unligated vessels , and vessels ligated for 48 hrs , 8 days , or 14 days . rt - qpcr analyses did not support significant mfap4 regulation within this period ( fig7 b ). highly intense immunostaining for α - sma was likewise found in the media . mfap4 deficiency significantly reduced ki - 67 , caspase 3 , and cd45 stained cells . ki - 67 stained proliferating cells were predominantly found in the neointimal area compared to the medial area ( approximately 3 : 1 in the mfap4 +/+ vessel ) ( fig7 a ), and there were no or few detectable ki - 67 - stained cells in the mfap4 −/− vessels at day 14 compared to mfap4 +/+ vessels ( 1 . 25 ± 0 . 48 ( sem ) cells / section versus 45 . 7 ± 24 . 0 ( sem ) ki - 67 - stained cells / section , respectively ( fig7 c ). likewise , mfap4 deficiency reduced the level of caspase 3 positive apoptotic cells and null cells were detected in the ligated mfap4 −/− vessel by day 14 ( fig7 c ). mfap4 deficiency further decreased the number of cd45 + cells in the vessel wall significantly . the counted number was on average 33 . 5 ± 10 . 0 ( sem ) cells / section in the mfap4 +/+ vessels and 2 . 75 ± 1 . 80 ( sem ) cells / section after 14 days ( fig7 c ). rt - qpcr analyses of integrin β 3 and mmp9 were performed in control vessels and vessels ligated for 48 hrs or 8 days ( for mmp9 ). the data did not support early or basal gene regulation of integrin β 3 . in contrast , the data supported that the arterial ligation lead to significant induction of the mmp9 gene product and that mfap4 deficiency reduced the expression . this reduction was evident 48 hrs after ligation , but disappeared during the prolonged healing response ( 8 days ) ( fig7 d ). deficiency of mfap4 alleviates allergic inflammation in murine acute model of asthma to examine whether mfap4 contributes to allergic asthma , mfap4 +/+ and mfap4 −/− littermates of the balb / c strain were subjected to acute ovalbumin ( ova )- induced allergic airway disease . leukocyte infiltration was checked in bronchoalveolar lavage ( bal ), and lung tissue was evaluated for signs of inflammation and airway remodeling . ova - treated mfap4 −/− mice exhibited significantly attenuated infiltration of neutrophils and eosinophils into the airway lumen . moreover , analysis of histological stainings revealed more prominent parenchymal inflammation and more pronounced early airway remodeling events in vvt mice , such as increased epithelial thickness and goblet cell hyperplasia . one mechanistic role for mfap4 is in integrin α v β 3 / 5 activation of vsmc adhesion , migration , and proliferation . in line with this , mfap4 deficiency delayed neointima formation after flow - cessation induced vascular injury . yet , the lack of mfap4 additionally reduced arterial outward remodeling and consequentially resulted in overall accelerated lumen reduction . other roles for mfap4 are as positive modulator of airway inflammation and airway remodeling . however , the mechanisms behind these functions remain hypothetic . well known integrin α v β 3 / 5 agonists often appear highly upregulated during vascular injury . in contrast to this , high vascular expression levels of mfap4 were evident before injury and during healing responses suggesting that mfap4 represent a novel integrin ligand with constitutive tissue expression . the systemic variation in mfap4 levels in symptomatic obstructive pad may thus primarily result from increased turnover of ecm . in vitro data generated in this study identifies vsmcs as sites of synthesis for mfap4 . the localization of human mfap4 to vsmcs combined with the observation that mfap4 binds the ecm fibrils supported a role for mfap4 in maintaining homeostatic functions in the vessel wall as known for other matricellular proteins and / or integrin α v β 3 ligands like osteopontin and vitronectin ( 29 , 36 , 37 ). the presence of integrin receptors for mfap4 in the vsmcs was following characterized . the utilised fetal cell line had a relatively high expression of integrin α v β 3 and therefore may represent partly dedifferentiated cells as commonly observed in ligated or otherwise injuried arteries . an alternatively tested adult haosmc line predominantly expressed integrin α v β 5 and interacted with mfap4 through this receptor ( data not shown ). the almost complete disruption of cellular adhesion onto immobilized mfap4 with blocking antibodies strongly indicates that integrin α v β 3 is the dominating mfap4 interaction partner in the present investigations . the relatively high level of mfap4 in the diseased as well as in the normal artery separates the expressional regulation of mfap4 from the common transient high expression of matricellular proteins and well - known integrin α v β 3 / 5 ligands and suggests that mfap4 mediated cellular effects primarily are regulated by other means than expression . the data further demonstrated that mfap4 induced functional distribution of integrins in focal adhesion sites as well as cellular migration , and proliferation . such in vitro observations are well known for integrin α v β 3 ligands osteopontin and vitronectin ( 38 , 39 ). the inventors observed reversal of the mfap4 induced cellular effects in the presence of mfap4 blocking antibodies and the observations indicated that growth factors like pdgf may determine the effect of mfap4 induced integrin activation . in order to study the effects of mfap4 on vsmc biology in vivo the mouse mfap4 gene was inactivated . histological examinations of tissues including arteries , skin and lung from unchallenged mfap4 −/− mice showed a normal gross appearance up till at least 3 months of age . the mean blood pressure did not differ between wild - type , and homozygous mfap4 −/− mice when measured through catheterization . blood pressure responses to phenylephrine infusions were normal in homozygous mfap4 −/− mice , indicating that the mfap4 gene deficiency did not alter the intrinsic pharmacological properties of smooth muscle cells in mice . thus , no relevant cardiovascular phenotype was found in the unchallenged mfap4 −/− mice . these observations supported that mfap4 is not essential for survival or normal cardiovascular development like for many other matricellular proteins including the integrin α v β 3 ligand osteopontin ( 30 ). in comparison , genetic ablation of integrin α v is demonstrated to be lethal ( 40 ) and integrin β 3 deficiency resulted in prolonged bleeding and decreased fetal survival ( 41 ) showing the roles of the integrins in a multitude of processes and in various cell types . mfap4 −/− mice underwent ligation of the left carotid artery in order to stop the blood flow and thereby causing the vessel to shrink in the luminal area due to the neointima formation and additional arterial remodeling ( 35 ). during the next 14 days , the eel of the ligated vessels in mfap4 −/− mice was reduced when compared to the unligated control vessels , without prominent acquisition of intimal mass . intravascular ultrasound has previously confirmed the presence of both outward and constricting remodeling after angioplasty suggesting that an increase in the total eel confined area is adaptive , whereas a decrease in the eel area contributes to restenosis with occlusion of the lumen ( 42 ). as the mfap4 −/− mice did not appear with prominent outward arterial remodeling , neither 14 nor 28 days after ligation , the delayed neointima formation ultimately resulted in a narrowing of the lumen 28 days after ligation . the effects appeared to be intrinsic consequences of the mfap4 −/− phenotype and were detectable between mouse strains with two different genetic backgrounds ( c57bl / 6n and balb / c ). reduced carotid neointima formation is previously observed in integrin β 3 deficiency ( 43 ) and with experiments performed using gene - deficiency for the integrin α v β 3 ligand vitronectin ( 44 ) or osteopontin inhibition experiments ( 45 ) where the gene deficiency appeared protective . mfap4 overexpression was recently demonstrated to preserve mmp ( collagenase degrading mmp1 and elastolytic mmp12 ) activity after photodamaging of skin , and was suggested to affect the synthesis of these mmps ( 34 ). mmp9 deficiency is demonstrated to reduce the vsmc migration and neointima formation in both endovascular denudation and carotid ligation experiments ( 46 , 47 ). a vein - graft model has further demonstrated that the expansive vessel remodelling may be induced in mmp9 deficient mice due to compensatory upregulation of mmp2 ( 48 ). the inventors &# 39 ; observations on mmp9 expression indicated that mfap4 deficiency reduces the synthesis of this enzyme . the transient and early reduction of the mmp9 transcription was not associated to outer vessel diameter expansion in the present studies , which rather showed constrictive remodeling with mfap4 deficiency . these data suggests that unrecognized extracellular proteases in addition to mmp9 may be reduced by mfap4 deficiency . as known for integrin α v β 3 - blocking antibodies ( 17 ) and other integrin α v β 3 antagonists ( 45 ) the mfap4 blocking antibodies may be anticipated to target vasculoproliferative processes including vsmc driven restenosis and neovascularization . one putative advantage with mfap4 blocking antibodies could be the selective inhibition of cellular integrins engaged in complexes with mfap4 , and the possible reduction of side effects from the integrin inhibition . moreover , prophylactic anti - mfap4 treatment could be initiated prior to an expected vascular damage due to the constitutive presence of mfap4 in the vessels . however , such treatment may require relatively high amounts of antibody , unless applied topically . moreover , although concern has existed regarding the safety of long - term systemic administration of integrin α v β 3 antagonists , including inhibition of wound healing and promotion of paradoxical cancerous activity ( 49 ) recent evidence has lessened this concern . integrin α v β 3 antagonism appears with an acceptable level of adverse effects ( 50 - 52 ), and sustained systemic exposure with integrin α v β 3 - or α v β 5 - blocking antibodies did not inhibit wound healing in monkeys and humans ( 53 , 54 ). in summary , the results of this study show that mfap4 plays a surprisingly multifacetted role in the vascular stenotic responses by promoting protective outward vessel remodeling but also the cellular growth and migration leading to hyperplasia . mfap4 is constitutively expressed and thus has the potential to serve as prophylactic terapeutical target for inhibition of vsmc growth and migration . additional obtained results show that absence of mfap4 attenuates ova - induced allergic airway disease . it indicates that mfap4 may serve as a therapeutic target in the treatment of allergic asthma . wild - type rmfap4 and different genetically modified versions of the protein was performed as previously described ( 7 ). c57bl / 6 / n mfap4 deficient mice were immunized for the production of monoclonal antibodies ( hg hyb 7 - 5 , 7 - 14 , and 7 - 18 ) against rmfap4 . freeze sections from a human vein with intimal hyperplasia was obtained from the vascular research unit , viborg hospital . formalin fixed normal human tissue was obtained from the tissue bank at the department of pathology , odense university hospital ( odense , denmark ). the local ethical committee in odense approved the use of the human tissue sections ( ref . no . vf20050070 ). mouse tissue was obtained from mfap4 −/− or mfap4 +/+ mice . utilised antibodies included ; anti - mfap4 ( hg - hyb 7 - 14 ), fluorescein isothiocyanate ( fitc )- anti - mfap4 ( hg - hyb 7 - 14 ), anti - α - sma ( dako # m0851 ), fitc - anti - α - sma ( sigma , clone 1a4 ), anti - integrin α v β 3 ( santa cruz # sc - 7312 ), anti - human cd45 ( roche # 760 - 4279 ), anti - mouse cd45 ( bd pharmingen , clone 30 - f11 ), anti - ki - 67 ( dako , clone mib - 1 ), anti - caspase - 3 ( cell signaling # 9664 ), and anti - fitc antibody ( p5100 , dako ). insoluble type i collagen from bovine achilles tendon and insoluble elastin from bovine aorta were supplied by sigma ( st . louis , mo ., usa ) and elastin products company , inc . ( owensville , mo ., usa ), respectively . five milligram of collagen or elastin was hydrated overnight in 10 mm tris buffered saline ( tbs ) 0 . 05 % ( w / w ) tween 20 , and 5 mm cacl 2 ( tbs / tw - ca 2 + ) or 10 mm ethylenediaminetetraacetic acid ( edta ) ( tbs / tw - edta ) at 4 ° c . and mixed with rmfap4 in tbs / tw - ca 2 + or tbs / tw - edta . after incubation at room temperature for 1 h , the water phase was recovered by centrifugation and analyzed by elisa . sandwich elisa assays were performed in 96 - well maxisorb microplates ( nunc ) essentially as described in molleken et al . 2009 ( 32 ). statistical significance between groups in in vitro and in vivo experiments was assessed by one - way or ( paired or unpaired ) two - way anova with bonferroni adjusted t - tests when relevant . data were analyzed using graphpad prism 5 . p & lt ; 0 . 05 was considered statistically significant . cells were grown at 37 ° c . in 5 % co 2 humidified incubator ( hera cell , heraeus ). fhaosmc &# 39 ; s or adult cells ( cell application , inc .) derived from normal human tunica intima and media of either fetal or adult aorta , were cultured in a smooth muscle cell growth medium ( cell application , inc ), or when allowed to differentiate in a smooth muscle cell differentiating medium ( cell application , inc .). cells were used in passages 3 - 7 . fixed and permeabilized cells were stained for 1 h at room temperature using 10 μg / ml fitc - anti - mfap4 in phosphate buffered saline ( pbs )/ bsa containing 0 . 2 % saponin ( w / w ). black 96 - well maxisorp fluoronunc ™ microtiter plates ( nunc ) were basically coated as above . in blocking experiments well were further incubated with 20 μg / ml of mfap4 blocking antibodies hg - hyb 7 - 5 , 7 - 14 or 7 - 18 or fhaosmcs were pre - incubated with either 25 - 100 μg / ml synthetic grgds or sdgrg peptides ( sigma - aldrich ) or 10 μg / ml anti - integrin antibodies ; anti - integrin α v , monoclonal mouse anti - human antibody clone l230 ( alexis biochemicals ); anti - integrin β 1 , monoclonal mouse anti - human antibody clone p4c10 ( millipore ); anti - integrin α v / β 5 , monoclonal mouse anti - human antibody clone p1 f6 ( santa cruz biotechnologies ); anti - integrin α v / β 3 , monoclonal mouse anti - human antibody clone lm609 ( millipore ); monoclonal mouse anti - fibrinogen c domain - containing protein 1 ( anti - fibcd1 ) antibody clone 12 - 5 ( control antibody produced in - house ( 55 )). a vybrant ™ cell adhesion assay kit ( molecular probes , invitrogen ) was used . the migration assay was performed using the oris ™ migration assembly kit ( platypus technologies madison , wis .) with coating as above . fhaosmcs were serum - starved before the addition of 0 . 5 % ( w / w ) fetal calf serum and 5 ng / ml pdgf - bb allowing cell migration . some well were incubated with anti - mfap4 antibody clones . migrated cells were detected using 4 ′, 6 - diamidino - 2 - phenylindole ( dapi ) solution ( invitrogen ). fhaosmcs were serum starved before seeding onto immobilized rmfap4 or fibronectin . blocking experiments were performed by incubating the protein coated wells with 20 μg / ml anti - mfap4 antibodies , or by preincubating suspended fhaosmcs with anti - integrin antibody in the presence of 0 . 3 % ( w / w ) fetal calf serum ± 5 ng / ml recombinant human pdgf - bb . the number of viable cells was following determined using an mtt - assay . sds - page and western blotting were performed using standard methods . primary antibodies included ; anti - α v ( cd51 ), monoclonal mouse igg clone 21 ( bd biosciences ); anti - β 1 , monoclonal mouse igg clone bv7 ( abcam ); anti - β 3 , polyclonal goat igg clone c - 20 ( santa cruz biotechnology ); anti - β 5 , polyclonal rabbit igg clone h - 96 ( santa cruz biotechnology ); anti - mfap4 , monoclonal hg - hyb 7 - 5 ; anti - glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ), monoclonal mouse igg clone 6c5 ( santa cruz biotechnology ); anti - α - sma , monoclonal mouse igg clone 1a4 ( sigma - aldrich ). secondary antibodies included : horseraddish peroxidase ( hrp )- labelled donkey anti - goat immunoglobulin ( santa cruz biotechnology ), goat anti - rabbit immunoglobulin hrp - labelled ( dako ), and rabbit anti - mouse immunoglobulin hrp - labelled ( dako ). pelleted fhaosmcs were resuspended with relevant primary anti - integrin antibodies described under “ cell adhesion assay ” or isotype matched anti - chicken ovalbumin ( the state serum institute , copenhagen ) and polyclonal anti - mouse fitc - conjugated goat f ( ab ′) 2 ( dako ) as secondary antibody . cells were analyzed using a becton dickinson ( bd ) flow cytometry facscan ™ ( bd biosciences ) and bd cell quest ™ software ( bd biosciences ). a targeting vector was constructed to delete genomic regions encompassing the core promoter region , exons 1 , 2 and a part of 3 for elimination of the mfap4 transcription . a neomycin expression cassette was ligated into the targeting vector . cj7 embryonic stem cells were electroporated with the linerized targeting vector . chimeric mice with the targeted es cell clones were developed and their descendants were backcrossed to c57bi / 6n and balb / c ( charles river laboratories international ) for 11 generations and maintained as heterozygotes . all mouse experimens were performed under a license obtained from the national animal experiments inspectorate who also approved the study ( ref . no . 2012 - 15 - 2934 - 00095 ). the arterial ligation model was essentially performed as described in kumar and lindner 1997 ( 35 ). mfap4 wt and ko mice were sensitized intraperitoneally with 20 ug ova with 2 mg alum in 200 ul pbs on days 0 and 7 . one week later mice were challenged intranasally with 20 ug ova in 50 ul pbs during three consecutive days . 2 mg alum in pbs and pbs only served as controls for sensitization and challenge , respectively . anaesthetized animals were sacrificed 24 h after final challenge . the trachea was cannulated , and bal was collected by washing the airway lumen four times with 0 . 5 ml pbs . cells were cytospun at 200 g for 5 min . and subsequently stained with hemacolor ( merck ). differential cell count was performed based on morphological criteria . lungs were excised , inflated with 10 % formalin and processed for histology . the level of parenchymal inflammation was assessed on slides stained with hematoxylin - eosin ( h - e ). periodic acid - schiff ( pas ) staining was used to visualise mucus - producing goblet cells . epithelial thickness was measured using imagej software . all analyses were performed in a blinded manner . total rna was from ligated and unligated carotid arteries was processed using standard methods relative expression was assessed using taqman ® assays ( applied biosystems by life technologies ); mfap4 : mm00840681_m1 ; integrin β 3 ( itgb3 ): mm004439980_m1 ; mmp9 : mm00442991_m1 ; tata - binding protein ( tbp ) ( endogenous control1 ): mm00446973_m1 ; gapdh ( endogenous control2 ): mm99999915_g1 . microfibrillar associated protein 4 ( mfap4 ) as modulator of the asm - dependent asthmatic remodelling . the present inventors originally identified mfap4 from lung washings [ 56 ] and subsequently localized the protein to various elastic tissues [ 57 ]. mfap4 is synthesized by and secreted from smooth muscle cells and is localized on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue [ 58 ]. mfap4 is a polymeric protein formed from 66 kda protein dimers [ 56 ]. the c - terminal fibrinogen - like domain is responsible for elastin and collagen binding , whereas the n - terminal region includes an rgd sequence responsible for interactions with integrin receptors [ 62 ]. proteome analysis of fibrotic liver tissue coupled with our measurements of serum mfap4 revealed that mfap4 serves as systemic marker for the fibrotic stages [ 63 ]. the inventors have produced mfap4 - deficient mice ( mfap4 −/−). high expression in the vascular compartment and intense staining for mfap4 in the vessel wall elastic fibres [ 64 ] has prompted us to investigate vascular remodelling in mfap4 −/− mice . using the carotid ligation model we have clear evidence that smooth muscle cell neointima formation and vascular smooth muscle cell ( vsmc ) proliferation are substantially reduced in the gene deficient mice . the present inventors could explain the observation through the interaction between mfap4 and integrins and have further demonstrated that vsmc migration and proliferation is significantly increased by mfap4 and may be reversed using our in house produced mfap4 - blocking antibodies . the putative role of mfap4 in asthma is unknown . however , our characterization of mfap4 as an integrin ligand , mediator of smooth muscle cell proliferation , adhesion and migration and the high pulmonary expression of mfap4 clearly suggest us to investigate its potential role in the asthma pathogenesis . the inventors divided female balb / c mice into 2 * 4 groups 1 ) mfap4 +/+ saline , 2 ) mfap4 +/+ house dust mite extract inhalation for seven weeks or ovalbumin sensitization and challenge for three weeks ( hdm or ova ), 3 ) mfap4 −/− saline , and 4 ) mfap4 −/− hdm or ova . morphometric analysis of the peribronchiolar smooth muscle layer showed increased asm layer thickness in mfap4 +/+ relative to mfap4 −/− ( fig9 a - b ). likewise , the collagen ( trichrome )- stained area per μm of basal membrane or the total hydroxyproline content was increased in the mfap4 +/+ mice ( fig9 c - d ). the central airway resistance was increased with allergic asthma but to a significantly higher degree in mfap4 −/− relative to mfap4 +/+ and there was likewise a tendency for increased metacholine induced airway hyperreactivity ( fig9 e - g ). moreover , the kcl - induced contraction force in the isolated tracheas measured after allergen treatment was increased in mfap4 - deficient mice ( fig9 h ). the bronchoalveolar lavage ( bal ) infiltration was significantly higher in ova - treated mfap4 +/+ mice relative to mfap4 −/− mice , and the same tendency was found in hdm - treated animals ( fig1 a - b ). infiltrating cells in lung tissue as well as in bal consisted primarily of eosinophils , whose numbers where significantly increased in mfap4 +/+ bal relative to mfap4 −/− ( fig1 c - d ). mfap4 levels measured by elisa as previously described [ 64 ] in bal and serum showed that allergic asthma results in increased mfap4 levels in these body fluids ( fig1 e - f ). mfap4 was located to the basal layer of the bronchi ( fig1 g ) and did not appear with visually detectable diseased induced changes in expression at that location . the inventors have contemplated a study based on perkins et al ., 2011 [ 66 ] and their own experience with similar assays using vascular smooth muscle cells . the following four prophetic experiments will detail the study . mfap4 - dependent integrin modulation of asm phenotype in vitro and ex vivo : primary human bronchial smooth muscle cells will be used as well as smooth muscle cells isolated from wildtype and mfap4 −/− mouse tracheas [ 66 ]. the present inventors will conclusively identify the specific integrin / s interacting with mfap4 in the asm . affinity purification of mfap4 ligands on mfap4 coupled to sepharose 4b will be performed using an array of relevant monoclonal anti - integrin antibodies . moreover , the present inventors will immunoprecipitate asm cell lysates with mfap4 antibodies and analyse by western blotting with the relevant integrin antibodies . the role of mfap4 and mfap4 - blocking antibodies in asm proliferation and migration : the present inventors will test mfap4 - dependent modulation of asm adhesion , proliferation and migration . to test if mfap4 treatment leads to conversion of quiescent asm into a more adhesive or proliferative state , recombinant mfap4 will be coated onto the tissue culture surface and / or platelet derived growth factor ( pdgf ) will be added into the culture medium and the proliferating fraction of cells will be estimated by colorimetric assay ( mtt assay ). the migratory response will be examined using similar cell culture conditions in a two - dimensional migration assay ( oris cell migration assembly kit , platypus technologies ). the integrin dependency in all assays will be tested using relevant integrin blocking antibodies and anti - mfap4 blocking antibodies . to test potential mfap4 - dependent eosinophil migration , the present inventors will isolate eosinophils from mfap4 −/− ( control ) and mfap4 +/+ mice sensitized and challenged with allergen from the lungs . the chemotactic response to increased concentrations of mfap4 will be checked by transwell assay . moreover , primary human bronchial smooth muscle cells will be grown on mfap4 - coated plates or in the presence of soluble mfap4 , and the production of eosinophil chemoattractants ccl11 , ccl24 and ccl5 will be analysed by qpcr and / or elisa . the present inventors will measure isometric tension in tracheal rings from mfap4 −/− ( control ) and mfap4 +/+ animals in the presence of mfap4 - blocking antibodies . tracheas will be isolated from animals and single open - ring , epithelium - denuded preparations will be mounted in organ baths . the tissue will be preincubated with antibodies and precontracted with kcl and following maximal relaxation will be established by the addition of isoproterenol . stepwise increasing concentrations of kcl or methacholine will be included to measure maximal tension . according to the outcome of analysis the experiment will be repeated on isolated bronchi from freshly resected cancer patient lung tissue in collaborative effort with thorax surgeon peter licht , odense university hospital . 1 . ross r . cell biology of atherosclerosis . annu rev physiol . 1995 ; 57 : 791 - 804 . epub 1995 / 01 / 01 . 2 . casscells w . migration of smooth muscle and endothelial cells . critical events in restenosis . circulation . 1992 ; 86 ( 3 ): 723 - 9 . epub 1992 / 09 / 01 . 3 . libby p , tanaka h . the molecular bases of restenosis . progress in cardiovascular diseases . 1997 ; 40 ( 2 ): 97 - 106 . epub 1997 / 11 / 05 . 4 . owens g k , kumar m s , wamhoff b r . molecular regulation of vascular smooth muscle cell differentiation in development and disease . physiol rev . 2004 ; 84 ( 3 ): 767 - 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