Patent Application: US-201515302288-A

Abstract:
an inhibitor of wnt / β - catenin signaling for use in the treatment and / or prevention of a pathology having vascular malformation is provided . the inhibitor may be a small molecule , a protein , a peptide or an antisense nucleic acid . the invention also relates to pharmaceutical compositions and to method of treatment .

Description:
the ccm3 - flox / flox mice were generated at taconicartemis ( koeln , germany ). two p - lox sequences were inserted that flank exons 4 and 5 of the murine ccm3 gene , to produce a loss - of - function mutation after excision by ore recombinase . these ccm3 - flox / flox mice were bred with cdh5 ( pac )- creert2 mice ( wang et al , 2010 ) for tamoxifen - inducible endothelial - cell - specific expression of ore - recombinase and ccm3 gene recombination . the ccm3 - flox / flox - cdh5 ( pac )- creert2 mice were further bred with bat - gal mice ( maretto et al , 2003 ) to monitor the activation of b - catenin transcription signaling , and with rosa 26 - enhanced green fluorescent protein ( eyfp ) ( rosa26eyfp ) mice ( srinivas et al , 2001 ) to monitor the expression of ore - recombinase through the expression of eyfp . tamoxifen ( sigma ) was dissolved in corn oil and 10 % ethanol ( at 10 mg / ml ), and then diluted 1 : 5 in corn oil before single intragastric administration to dpn 1 - 2 pups ( 35 mg / kg body weight ), as described in 14 . the control ( wild - type ) mice included ccm3 - flox / flox - cdh5 ( pac )- creert2 - bat - gal mice treated with the vehicle used to dissolve the tamoxifen ( corn oil plus 2 % ethanol ), and ccm3 +/+ - cdh5 ( pac )- creert2 - bat - gal mice treated with tamoxifen . ccm1 - ecko and ccm2 - ecko have been obtained from ccm1 - flox / flox and ccm2 - flox / flox mice as described in details above for ccm3 - ecko mice , see also maddaluno et al , 2013 and boulday et al , 2011 . the following probes were used for the mouse genotyping : wild - type ccm3 allele : 5 ′ gat agg aat tat tao tgc cct tcc 3 ′ ( seq id no . 1 ), 5 ′ gao aag aaa gca ctg ttg acc 3 ′ ( seq id no . 2 ); deleted ccm3 gene after recombination induced by cre recombinase : 5 ′ gat agg aat tat tao tgc cct tcc 3 ′ ( seq id no . 3 ), 5 ′ gct acc aat cag ctt ctt agc cc 3 ′ ( seq id no . 4 ), cdh5 ( pac )- creert2 gene : 5 ′ cca aaa ttt gcc tgc att acc ggt cga tgc 3 ′ ( seq id no . 5 ), 5 ′ atc cag gtt acg gat ata gt 3 ′ ( seq id no . 6 ); bat - gal gene : 5 ′ cgg tga tgg tgc tgc gtt gga 3 ′ ( seq id no . 7 ), 5 ′ acc acc gca cga tag aga ttc 3 ′ ( seq id no . 8 ); rosa 26 eyfp gene : 5 ′ gcg aag agt ttg tcc tca acc 3 ′ ( seq id no . 9 ), 5 ′ gga gcg gga gaa atg gat atg 3 ′ ( seq id no . 10 ), 5 ′ aaa gtc gct ctg agt tgt tat 3 ′ ( seq id no . 11 ). sulindac sulfide ( sigma ) and sulindac sulfone ( sigma ) were both dissolved in dmso and further diluted 1 : 50 in corn oil . they were administered intragastrically , daily ( 30 mg / kg body weight ), starting one day after the induction of recombination . the control mice were treated in parallel with vehicle only ( corn oil plus 2 % dmso ). the inventors did not observe either increased bleeding from vascular lesions or mortality in drug - treated ccm3 - ecko mice in comparison to vehicle - treated ones . drugs were added to confluent cells for 48 h before the indicated assays . final concentrations for iwp2 and iwp12 , both from sigma - aldrich , were : 0 . 5 , 2 , 5 dm . drugs were dissolved in dmso , control treatment ( vehicle ) was 0 . 1 % dmso final concentration , as for drug treatment . murine recombinant dkk1 ( r & amp ; d ) was 0 . 5 dm on cells . murine recombinant wnt3a ( r & amp ; d ) was 5 ng / ml for the time indicated in the figure legends . in vitro isolation , culture and recombination of endothelial cells from the ccm3 - flox / flox mice endothelial cells from the ccm3 - flox / flox mice ( 8 - 10 weeks old ) were isolated from the brain as previously described 13 . recombination of the floxed ccm3 gene was induced by treating the cells at culture day 1 with the adenocre viral vector , as previously described 59 . the control endothelial cells were an aliquot of the same endothelial cell preparation treated with adenogfp , instead of adenocre . the cells were then maintained in culture for up to a further 7 days before analysis , as described in the main text . drug treatments were for 48 h before the processing of the cells . in some experiments , endothelial cell lines from the lungs of ccm3 - flox / flox mice ( 8 - 10 weeks old ) were immortalized in culture through retroviral expression of polyoma middle t gene 60 . ablation of the ccm3 gene was achieved with the adenocre viral vector ( with adenogfp in the control cells ). these cells were then maintained in culture for up to 25 passages without detectable changes in the effects of this ccm3 ablation . these endothelial cell lines responded to the absence of ccm3 in a comparable way to both primary cultures of brain endothelial cells in vitro and brain endothelial cells in vivo . drugs were added to confluent cells for 48 h before the indicated assays . the final concentrations used were : 135 mm sulindac sulfide , 125 mm sulindac sulfone , 200 mm silibinin , 40 mm curcumin , 40 mm resveratrol , and 250 mm salinomycin ( all from sigma - aldrich ). as all of these were dissolved in dmso , control treatment ( vehicle ) was 0 . 1 % dmso final concentration , as for drug treatment . immuno - staining for fluorescence microscopy of brain sections , retinas and cells in culture brains and eyes from mice pups were fixed in 3 % paraformaldehyde immediately after dissection , and this fixing was continued overnight at 4 ° c . the retinas were dissected from the eyes just before staining as the whole mount . fixed brains were embedded in 4 % low - melting - point agarose and sectioned along the sagittal axis ( 150 mm ) using a vibratome ( 1000 plus , the vibratome company , st . louis , mo ., us ). brain sections and retinas were stained as floating samples in 12 - well and 96 - well plates , respectively . they were blocked overnight at 4 ° c . in 1 % fish - skin gelatin with 0 . 5 % triton x100 and 5 % donkey serum in phosphate - buffered saline ( pbs ) containing 0 . 01 % thimerosal . the samples were incubated overnight at 4 ° c . with the primary antibodies diluted in 1 % fish - skin gelatin with 0 . 25 % triton x100 in pbs containing 0 . 01 % thimerosal . following washing with 0 . 1 % triton x100 in pbs , the secondary antibodies were added for 4 h at room temperature in 1 % fish - skin gelatin with 0 . 25 % triton x100 in pbs containing 0 . 01 % thimerosal . the incubation with dapi was in pbs for 4 h , which was followed by several washes in pbs , post - fixating with 3 % paraformaldehyde for 5 min at room temperature , and further washes in pbs . the brain sections were mounted in vectashield with dapi , and the coverslips fixed with nail varnish ; the retinas were mounted in prolong gold with dapi . cells cultured in vitro were fixed and stained as described previously 46 . the following antibodies were used : anti - pecam ( hamster ; mab1398z , millipore ); anti - b - galactosidase ( chicken ; ab9361 , abcam ); anti - ve - cadherin ( rat monoclonal ; 550548 , bd biosciences ); anti - ve - cadherin ( goat ; sc - 6458 , santa cruz ); anti - active - b - catenin ( mouse monoclonal clone 8e7 ) dephosphorylated on ser37 and thr41 , millipore ); anti - total - b - catenin ( mouse monoclonal ; cell signaling ); anti - s100a4 ( rabbit ; 07 - 2274 , millipore ); anti - klf4 ( goat ; af3158 , r & amp ; d ); anti - cd44 ( rat ; 553131 , bd biosciences ); anti - id1 ( rabbit ; sc - 488 , santa cruz ); anti - asma ( mouse monoclonal ; f3777 , sigma ); anti - gfp ( rabbit ; a - 6455 , invitrogen ); anti - podocalyxin ( goat ; af1556 , r & amp ; d ); anti - phospho - histone h3 ( rabbit ; ab51776 , millipore ); anti - ccm3 ( rabbit ; eurogentec ); anti - p - smad1 ( rabbit ; 9516 , cell signaling ); anti - endomucin ( rabbit ; sc - 65495 , santa cruz ); anti - α - tubulin ( mouse monoclonal ; t9026 , sigma ). biotin - conjugated isolectin b4 ( vector lab ), revealed with alexa555 - conjugated streptavidin ( molecular probes ), was also used to identify endothelial cells in retina and brain sections . the secondary antibodies for immunofluorescence were anti - alexa448 and anti - alexa555 , and cy3 - conjugated antibodies raised in the donkey against immunoglobulin of the appropriate animal species ( molecular probes or jackson laboratories ). the secondary antibodies for western blotting were hrp - linked anti - mouse , anti - rat and anti - rabbit antibodies ( cell signaling ), and hrp - linked anti - goat antibodies ( promega ) rna extraction was performed with rneasy kits ( 74106 ; promega ). the rna ( 1 μg ) was reverse transcribed with random hexamers ( high capacity cdna archive kits ; applied biosystems ). the cdna was amplified with taqman gene expression assays ( applied biosystems ) using a 7900 ht thermocycler ( abi / prism ). for each sample , the expression levels were determined with the comparative threshold cycle ( ct ) method , and normalized to the housekeeping genes encoding 18s and glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ). for amplification , the following probes ( applied biosystems ) that have been validated to recognize the mouse transcripts in rtpcr were used : axin2 , nkd1 , lef1 , ccnd1 , cmyc - klf4 , ly6a , s100a4 , id1 , cdh2 , acta2 , cd44 . the probes to identify the ccm3 mrna transcript were custom designed , as : forward , cgagtccctccttcgtatgg ( seq id no . 12 ); reverse , gctctggccgctcaatca ( seq id no . 13 ); reporter sequence , ctgatgacgtagaagagtaca ( seq id no . 14 ). for the detection of b - catenin - dependent transcription of a reporter target , the top - flash plasmid was used ( 0 . 3 mg / cm 2 cell culture area ), which contains seven tcf / lef binding sites that control the transcription of firefly luciferase ( lluis et al , 2008 ). this was transfected into the endothelial cells from lung using lipofectamine 2000 , according to the manufacturer instructions ( invitrogen ). the pcmv plasmid for constitutive expression of b - gal was co - transfected ( 0 . 1 mg / cm 2 ), for normalization of luciferase expression over transfection efficiency . as the negative control , a fop - flash plasmid was used that contained six mutated ( i . e ., inactive ) tcf / lef sites upstream of a minimal promoter and the firefly luciferase gene ( 0 . 3 mg / cm 2 ). this was co - transfected with the b - gal plasmid , for normalization , as above . the dual - light reporter gene assay system ( applied biosystems ) for the combined detection of firefly luciferase and b - gal was used . the cell extraction and detection of chemiluminescence ( glomax 96 microplate luminometer ; promega ) was carried out according to the manufacture instructions . standard procedures were used to extract and analyze the protein content by western blotting and immunoprecipitation 46 . nuclear fractionation was as described previously 62 . for the classification and counting of lesions , entire brains from dpn 9 littermate pups were sectioned and immunostained for pecam as described above and in the methods . sections were then examined under wide - field fluorescence microscopy ( 10 × and 20 ×). lesions were classified as described in 63 as mulberry ( multiple cavernae , group of more than two contiguous cavernae ), single caverna ( single dilated vessel with maximal diameter accommodating more than 25 red blood cells ), or telangiectases , tortuous small vessels with abnormally dilated lumen ). the total numbers of lesions were calculated by summing all of the types of lesions . as the sections were 150 - mm thick , a correction was applied to the number of mulberry lesions , which can span two sections . therefore , the number of mulberry lesions was divided by 2 . 5 . the lesions were counted and classified independently by two observers who were blinded to the treatments . the maximal diameter of the mulberry lesions and single cavernae was used for statistical comparison . ve - cadherin expression was silenced using sirna oligos to murine ve - cadherin ( smart pool , thermo scientific ; target sequences : agacagaccccaaacguaa ( seq id no . 15 ), gaaaauggcuugucgaauu ( seq id no . 16 ); agggaaacaucuauaacga ( seq id no . 17 ); ccgccaacaucacggucaa ( seq id no . 18 )), respectively , and lipofectamine 2000 for transduction as described in lampugnani et al , 2010 . non - parametric wilcoxon signed - rank tests were used to determine the statistical significance of the lesion burdens after the pharmacological treatments in vivo . student &# 39 ; s two - tailed non - paired t - tests were used to determine the statistical significance in the other in vitro and in - vivo analyses . the significance level was set at p & lt ; 0 . 05 . transcription activity of b - catenin is enhanced in vivo in endothelial cells of endothelial - cell - specific ccm3 - knockout mice the in vivo mouse system presented here was initially generated through the cross of ccm3 - floxed / floxed mice with cdh5 ( pac )- creert2 mice 14 , for tamoxifen - inducible endothelial - cell - specific expression of cre - recombinase and ccm3 gene recombination . these mice were then further crossed with bat - gal mice 15 , which show b - catenin - activated expression of the nuclear b - galactosidase ( b - gal ) reporter gene . as previously reported for ccm2 6 , these mice with endothelial - specific - inactivation of ccm3 gene induced postnatally ( ccm3 - ecko ) presented marked malformations and hemorrhages in the brain and retina vasculature comparable to ccm vascular lesions in patients . this was previously reported for ccm2 in an identical murine model 6 and for ccm2 and ccm3 in a distinct murine system 7 . as ccm3 ecko mice develop vascular malformations in the central nervous system this model provides a tool for testing pharmacological treatments as described below . for details of this experimental model , see methods and fig1 . here , b - catenin - dependent transcription of the nuclear b - gal reporter gene is increased in vivo in endothelial cells of brain vessels of the newborn ccm3 - ecko , in comparison to matched control animals . as illustrated in the immunostaining in fig1 a and 1 b , the quantification by random - field counting using pecam labeling of endothelial cells saw the b - gal - positive nuclei ( 0 . 86 ± 0 . 15 positive nuclei per field ; 7 . 2 % positive nuclei of total 600 endothelial nuclei scored ) of the control brain endothelial cells from the wild - type bat - gal mice significantly increased by 4 - fold in the ccm3 - ecko brain endothelial cells ( 5 . 0 ± 1 . 7 positive nuclei per field ; 36 % positive nuclei of total 700 endothelial nuclei scored , p & lt ; 0 . 05 ; t - test ) ( dpn 9 littermate pups ). in these ccm3 - ecko brain endothelial cells , the b - gal - positive nuclei were distributed both in established caverns and in telangiectases ( 67 % b - gal - positive endothelial nuclei in vascular lesions ) and in pseudo - normal vessels ( 15 % b - gal - positive endothelial nuclei in pseudo - normal vessels ). expression of b - gal was also observed in endothelial cells of the retina of the ccm3 - ecko mice , in comparison with those from the control wild - type bat - gal mice ( fig1 c ). endothelial cells isolated from the brain of the ccm3 - floxed / floxed mice ( fig2 a , wt , primary culture ) were compared with those after recombination of the ccm3 gene in vitro ( ccm3 - knockout brain endothelial cells ; see methods ) ( fig2 a , ko , primary culture ), which showed active b - catenin in the nucleus ( i . e ., dephosphorylated on ser37 and thr41 16 ). this effect paralleled strong alterations to the adherens junction organization that was seen as delocalization from the junctions of both active b - catenin ( fig2 a ) and ve - cadherin ( fig3 c , vehicle ). for the similar disorganization of ve - cadherin from endothelial junctions in vivo , see fig4 b , vehicle . due to limitations in both the supply of freshly isolated endothelial cells from the brain and their extremely limited mitotic index after the first in vitro passage detailed analyses of their b - catenin nuclear distribution and signaling were not possible . the inventors therefore established cultured endothelial cell lines where ccm3 was recombined in vitro as above 17 ( ccm3 - knockout endothelial cell line ) ( see methods ). in these ccm3 - knockout endothelial cell line , the inventors confirmed enhanced nuclear localization of b - catenin both by immunofluorescence ( fig2 b ) and cell fractionation ( fig2 c ). in addition , beta - catenin and tcf / lef - dependent transcription of the exogenous luciferase gene was measured in top / fop flash reporter assays . in these ccm3 - knockout endothelial line , the transcription activity of b - catenin was significantly increased by 2 . 7 - fold , compared to control wild - type cells (± 0 . 2 sd ; p & lt ; 0 . 01 ) ( fig1 d ). furthermore , the expression of some typical endogenous targets of b - catenin transcription activity was increased in these ccm3 - knockout endothelial line ( i . e ., axin2 , lef1 , ccnd1 18 ) and inhibited by in vitro infection with the adeno viral vector coding for a dominant - negative tcf4 17 ( fig2 d ). the inventors also analyzed the expression of genes related both to acquisition / maintenance of endothelial progenitor phenotype and endothelial - to - mesenchymal transition , ( endmt ) 20 , 21 , as b - catenin transcription signaling has been shown to regulate the processes of differentiation and epithelial - to - mesenchymal transition ( emt ) in other cell types 22 , 23 , 24 . in addition , endmt was found to play a critical role in the development of the vascular lesions in a murine model of endothelial - specific ccm1 knockout ( maddaluno , l . et al ., nature 498 ( 7455 ): 492 , 2013 ). transcription of klf4 25 , 26 , ly6a 27 , 28 and s100a4 29 , 20 , id1 30 , 31 , cdh2 20 , acta2 20 were found to be significantly enhanced in ccm3 - knockout endothelial cells as compared to control cells ( fig2 e , endothelial line and fig3 b , primary culture ). in addition , these increases were dependent on b - catenin transcriptional activity , as they were inhibited by dominant - negative tcf4 17 , as above , ( fig2 e ). the enhanced transcription of these genes also corresponded to their increased protein expression ( see vehicle - treated ccm3 - knockout in fig1 and 19 , endothelial line , and fig3 c , primary endothelial cells , and their up - regulation in vivo in vehicle - treated ccm3 - ecko brain endothelial cells in fig5 and fig2 ). thus , abrogation of ccm3 expression in endothelial cells leads to increase in b - catenin transcriptional activity and target gene expression both in vivo and in vitro . in the light of these data , the inventors investigated the effects of anti - b - catenin agents in vivo , on the brain vascular lesions of these ccm3 - ecko mice . sulindac sulfide reduces b - catenin transcription activity and expression of endothelial progenitor and endmt markers in endothelial cells from the ccm3 - ecko mice the inventors then initially tested a range of agents in their experimental model in vitro that have been described as affecting b - catenin signaling 32 and , most importantly , are already in clinical use : sulindac sulfide , sulindac sulfone 33 , 34 , silibinin 35 , curcumin 36 and resveratrol 37 , 38 . salinomycin 39 , a reported inhibitor of wnt receptor signaling , was also included , although its use to date has been limited to experimental models . sulindac sulfide and sulindac sulfone were the most effective of these for inhibition of expression of endogenous targets of b - catenin transcription activity ( see above ) in the ccm3 - knockout endothelial cell line ( fig1 a ). in addition , sulindac sulfide reduced b - catenin transcriptional activity as measured by the top / fop flash reporter assays ( fig1 d ). the inventors therefore further analyzed the effects of sulindac sulfide on the ccm3 null phenotype . in primary cultures of ccm3 - knockout brain endothelial cells , the inventors found that sulindac sulfide effectively inhibited the expression of endogenous b - catenin target genes ( fig3 a and see inhibition by dominant - negative tcf4 , as above , fig3 b ). in parallel , sulindac sulfide strongly inhibited the nuclear localization of active b - catenin while increasing its concentration at cell - cell junctions ( fig3 c ). in parallel , ve - cadherin was also more localized to cell - cell contacts in these cells ( fig3 c ) and in ccm3 - knockout endothelial cell line ( fig1 b ). consistently , by co - immunoprecipitation and western blotting analysis sulindac sulfide restored the reduced association between b - catenin and ve - cadherin ( minus 35 %± 0 . 32 sd , p & lt ; 0 . 05 ) in ccm3 - knockout endothelial line ( fig1 c ). in parallel with inhibition of transcription of b - catenin target genes , sulindac sulfide also inhibited the overexpression of respective proteins in ccm3 - knockout endothelial cells ( fig3 c , primary culture , and fig1 , endothelial cell line ). sulindac sulfide was then investigated in vivo in newborn mice after induction of ccm3 - ecko . the inventors found that treatment with sulindac sulfide inhibited the expression of nuclear reporter gene b - gal ( fig4 a ) and of b - catenin target genes ( fig5 ) in the endothelial cell of brain vasculature too . ve - cadherin also appeared to be better localized at endothelial cell - cell junctions in vivo in the brain vessels of newborn ccm3 - ecko mice treated with sulindac sulfide ( fig4 b ). sulindac sulfide reduces development of vascular lesions in the brain and retina of the ccm3 - ecko mice a crucial aspect of the present study is whether inhibition of b - catenin signaling by sulindac sulfide may also reduce the vascular lesions in ccm3 - ecko pups . the inventors found that , indeed , the mean number and dimension of vascular lesions were reduced by sulindac sulfide treatment . as illustrated in the immunostaining in fig6 a and quantified in fig6 b , the mean number (± sd ) of vascular lesions per brain in the vehicle - treated ccm3 - ecko pups was 166 . 8 ± 22 , with 72 . 6 ± 9 vascular lesions with sulindac sulfide treatment ( p & lt ; 0 . 005 ; non - parametric wilcoxon signed - rank test ) and the mean maximal diameter of mulberry lesions (± sd ) in the vehicle - treated ccm3 - ecko pups was 386 ± 56 mm and 244 ± 38 mm with sulindac sulfide treatment ( p & lt ; 0 . 05 , t - test ). sulindac sulfide treatment did not significantly reduce the maximal diameter of single cavernae . sulindac sulfide treatment also inhibited the vascular malformations in the retina of ccm3 - ecko mice . in these mice the retinas show multiple - lumen vascular lesions that are particularly concentrated at the periphery of the vascular network . such lesions develop from veins , which are enlarged , although straight ( compare vehicle for wt and ccm3 - ecko in fig6 c and 6 e and fig1 for the venous marker endomucin ). sulindac sulfide partially normalized this aberrant vascular network in ccm3 - ecko mice ( fig6 c and 6 d ). in addition , the enlargement of the most internal tract of the veins that characterizes the retinas of these ccm3 - ecko mice was inhibited after sulindac sulfide ( 89 . 5 ± 7 . 1 mm in vehicle - ecko versus 35 ± 7 . 8 mm in sulindac sulfide - ecko , mean ± sd of 30 measurements in 14 retinas each for wt and ko ) ( fig6 e and fig1 a and 18 b , for the effects of sulindac sulfide on vessel diameter and veins in the brain of ccm3 - ecko pups ). at variance , arteries do not show this aberrant phenotype ( fig6 c and 6 e ). the data reported above strongly suggest that sulindac sulfide may have a therapeutic activity in ccm patients . however , it has been reported that this drug inhibits cycloxygenease in platelets possibly increasing the risk of hemorrhage . the inventors therefore tested sulindac sulfone , which is devoid of anti - cyclooxygenase activity 33 and does not have an impact on coagulation response . as observed after sulindac sulfide treatment , sulindac sulfone also reduced the nuclear accumulation of active b - catenin and restored cell - cell junctions in cultured ccm3 - knockout endothelial cell line ( fig1 ). in addition , sulindac sulfone inhibited the expression of b - catenin target genes , ( see for instance klf4 and s100a4 in fig1 ), as did sulindac sulfide ( see above ). when tested in vivo , sulindac sulfone reduced the number of lesions in the brain of the ccm3 - ecko mice to a level comparable to sulindac sulfide ( the mean number of lesions per mouse brain (± sd ) in the untreated control was 153 . 5 ± 28 and was reduced to 68 . 6 ± 10 with sulindac sulfone treatment , p & lt ; 0 . 01 ; non - parametric wilcoxon signed - rank test ; fig2 a and 20 b ). in addition , in vivo , in the endothelium of brain vessels of the ccm3 - ecko mice , sulindac sulfone inhibited the expression of klf4 and s100a4 ( fig2 c ). similar results indicating that sulindac sulfone ( exisulind ) inhibits the formation of cavernoma lesions in the brain of ccm1 - ecko mice have also been obtained . kinetics of activation of βeta - catenin signaling in endothelial cells in brain cavernomas of ccm3 - ecko mice ccm3 ecko murine model is first choice in proof of principle experiments , in particular testing the inhibitory activity of a drug as it develops the most serious phenotype as also observed in patients . as reported in fig7 βeta - catenin signaling is early enhanced in the lesion and such activation precedes the activation of tgf - β / bmp pathway that the inventors have reported to contribute to the maintenance of the ccm phenotype ( maddaluno et al , 2013 ). the in - vivo mouse system used was generated through the cross of ccm3 - flox / flox mice with cdh5 ( pac )- creert2 mice , to obtain tamoxifen - inducible endothelial - cell - specific expression of cre - recombinase and ccm3 gene recombination ( ccm3 - ecko mice ). these mice were then crossed with bat - gal reporter mice ( 16 ), which show βeta - catenin - activated expression of nuclear β - galactosidase ( β - gal ). as reported in fig7 a ( upper panels ) the inventors could observe a significantly higher β - catenin transcription signal in the nuclei of endothelial cells in ccm3 - ecko mice in comparison to matched controls . this difference was detectable since early stages ( 3dpn ) after induction of ccm3 recombination ( at 1dpn ). in brain sections of ccm3 - ecko mice endothelial cells with j3 - gal - positive nuclei could be found both in pseudo - normal vessels and in cavernae of any size ( fig8 ). in contrast , phospho - smad1 ( p - smad1 ) staining , in separate sections ( fig7 a lower panels ) and in co - staining for β - gal ( fig7 b ) was not enhanced after ccm3 ablation in 3dpn pups , but it was increased in 9dpn pups ( fig7 c ). similar results were obtained for p - smad3 ( not shown ). p - smad1 was significantly high in middle - large size lesions only ( maximal diameter 50 μm in 9dpn pups ) ( fig8 ). this result strongly support the pharmacological targeting of β - catenin signaling for the inhibition of initiation of new lesions . the appearance of de - novo lesions is a specific feature of the familiar form of ccm in patients . in vitro data on regression of mutated phenotype using sulindac sulfide and sulindac sulfone to inhibit β - catenin signaling are shown in fig4 and fig1 . correlation between β - catenin signaling and expression of endmt markers analyzing in vivo the kinetics of the two processes in endothelial cells of brain cavernomas of ccm3 - ecko mice expression of stem - cell / endmt markers ( klf4 , ly6a , s100a4 and id1 ) was high in 3dpn ccm3 - ecko pups ( fig9 a - d , and e , single positive ) and was concentrated in endothelial cells with β - gal positive nuclei ( fig9 e , co - localization ). at 9dpn β - gal expression in endothelial cells of ccm3 - ecko pups decreased while stem - cell / endmt markers remained high ( fig9 e ). the role of β - catenin signaling in established lesions is under scrutiny , at the present as discussed in the previous paragraph a canonical target of activated β - catenin - driven transcription , axin2 , is indeed enhanced in endothelial cells in culture after ablation of ccm1 and ccm2 genes besides ccm3 ( fig1 ), as well as endmt markers . analyzing in details ccm1 ko endothelial cells in culture , the inventors observed nuclear localization of active β - catenin ( dephosphorylated on ser37 and thr41 and escaping proteosomal degradation fig1 a ), as already reported in ccm3 - ko endothelial cells ( fig2 ). in addition , such nuclear β - catenin is transcriptionally active as indicated by the two fold - increase expression of the exogenous luciferase gene as measured in top / fop flash reporter assays ( a measure of activation of β - catenin / tcf / lef - dependent transcription ; fig1 b ). most importantly , inhibition of β - catenin signaling using sulindac sulfone ( exisulind ) inhibits the expression of endmt markers in ccm1 ko endothelial cells in culture ( fig1 c ). these data supports the concept that mutation of any ccm gene induces β - catenin signaling in association to acquisition of de - differentiated phenotype ( expression of endmt markers , fig1 ). in addition , sulindac sulfone ( exisulind ) induces re - organization of endothelial cell - to - cell contacts in ccm1 ko endothelial cells in culture , as reported for ccm3 ko endothelial cells in fig3 . similar results were also obtained in ccm2 ko models . mechanism through which β - catenin signaling is activated in response to ccm ablation in endothelial cells activation of β - catenin - mediated transcription in ccm3 - knockout endothelial cells was cell - autonomous since : a ) it was observed in absence of exogenous wnt ; b ) the porcupine inhibitors iwp2 and iwp12 ( 26 , 27 ) that inhibit ligand production , as well as dkk1 , an inhibitor of ligand - receptor interaction that is a competitor of wnt co - receptor lrp5 / 6 ( 26 , 27 ), did not inhibit transcription of typical β - catenin ( fig1 a - d ); c ) lrp6 phosphorylation was not increased ( fig1 e and f ); d ) stimulation by exogenous wnt3a did not induce expression of stem - cell / endmt markers , while the constitutively active form of β - catenin , lef - δ13cta ( 28 ), did ( fig1 g and h ). taken together these data indicate that in ccm3 - knockout endothelial cells enhanced nuclear localization and transcription activity of β - catenin do not depend on a classical ligand - receptor interaction . the inventors reported previously that silencing or dismantling of ve - cadherin from endothelial junctions can up - regulate β - catenin signaling ( 18 ). consistently , the inventors observed here that silencing ve - cadherin by sirna activated the expression of endmt markers ( s100a4 and id1 ) besides typical β - catenin targets ( axin2 , ccnd1 and nkd1 , fig1 i ) and promoted nuclear localization of active β - catenin ( fig1 a ). on the contrary , ve - cadherin knockdown did not enhance the phosphorylation of smad1 ( fig1 b ). these data suggest that in ccm the first trigger of β - catenin signaling is the dismantling of ve - cadherin junctions that , in turn , causes the release of β - catenin in the cytoplasm and nuclear translocation . this process precedes and possibly contributes to the activation of tgf - β / bmp signaling for lesion progression . interestingly , dismantling of cell - to - cell junction in endothelial cells represents a common feature of the brain cavernomas induced by ablation of any ccm gene , as reported for ccm1 - ecko , ccm2 - ecko and ccm3 - ecko ( fig1 ). the inventors have previously reported disorganization of ve - cadherin in brain cavernomas of ccm1 patients ( lampugnani et al , 2010 ). therefore , disorganization of junctional ve - cadherin appears a general feature of endothelial cells lining vascular cavernous malformations both in murine models and in patients . this may be accompanied by dissociation of β - catenin from ve - cadherin with enhanced nuclear translocation and increased β - catenin driven transcription also in patients , as reported above in experimental murine models . at the present , a direct proof of the activation of β - catenin signaling in brain cavernomas in patients is very difficult due to technical limitations in detecting markers of such activation in human samples . in summary , these results support the use of anti - β - catenin compounds for treatment of pathologies characterized by vascular malformation , in particular ccm patients . here , the inventors report that endothelial - cell - selective deletion of the ccm3 gene activates b - catenin transcription signaling in vivo in brain endothelial cells . pharmacological inhibition of b - catenin transcriptional activity with the nsaids sulindac sulfide and sulindac sulfone reduces the number and dimension of cerebral and retinal vascular malformations in this murine model suggesting that b - catenin transcription signaling in endothelial cells contributes to the pathogenesis of ccm3 - mediated vascular lesions . ccm malformations develop largely , although not exclusively , in the central nervous systems in patients and in mouse models 6 , 8 . consistent with a critical role for deregulated b - catenin signaling in the endothelium in ccm pathology , the canonical wnt pathway is a well - established determinant for the specification of the phenotype of endothelial cells at the blood - brain barrier 11 - 13 . however , wnt signaling must be abrogated postnatally to avoid abnormal vascular proliferation and morphogenesis in the central nervous system 11 - 13 . in a murine model of endothelial specific b - catenin - gain - of - function the inventors observed vascular lesions in the retina comparable to those observed here in ccm3 - ecko 40 . in tumor cells , the sustained induction of canonic wnt signaling is associated to increased growth and invasion 18 , 24 . in particular , upon b - catenin - mediated transcriptional activation , carcinoma cells switch from an epithelial to a mesenchymal phenotype ( emt ) 18 , 24 . the inventors report here that ccm3 - knockout endothelial cells undergo a similar change in phenotype and upregulate a series of genes typical of emt / endmt 20 . thus , a reasonable hypothesis is that ccm lesions originate from uncontrolled kinetics and location of b - catenin signaling in endothelial cells of brain vessels . it has recently been reported in retina and brain endothelial cells that activation of the canonical wnt pathway by norrin / frizzled4 induces development and maintenance programs through both cell - autonomous and cell - nonautonomous signaling 41 . this can explain local vascular phenotypes in different regions of the central nervous system 41 . deregulated b - catenin signaling in ccm could be supported by either endothelial - autonomous activation of the wnt / b - catenin pathway or by abnormal responses of mutated endothelial cells to environmental wnt , or by a combination of the two . the inventors &# 39 ; data indicate that at least the first of these mechanisms appears to operate in endothelial cells in culture after ablation of the ccm3 gene . indeed , some b - catenin targets and progenitor / endmt markers are activated through b - catenin transcription signaling under basal conditions in the ccm3 - knockout endothelial cells , as their expression is inhibited by a dominant - negative tcf4 . glading and ginsberg 10 reported similar activation of b - catenin transcription activity in bovine aorta endothelial cells and primary human arterial endothelial cells in culture after depletion of ccm1 using rna interference . they also reported inhibition of b - catenin signaling by ccm1 in epithelial cells , both in vitro and in vivo . in addition , they reported that the phenotype of b - catenin - driven intestinal adenomas in apc min /+ mice is exacerbated in a ccm1 +/− background . this implies a more general regulatory role for ccm1 , and possibly ccm3 , in b - catenin signaling in other cells as well as in endothelial cells . as the occurrence of intestinal and other neoplasias in familial ccm patients does not appear to be increased , the specificity of vascular and organ localization for the ccm pathology suggests that endothelial differentiation and / or local factors in the organ cooperate with wnt / b - catenin signaling for the expression of the mutated phenotype 42 . in particular , as far as is known for ccm3 , mutation of this gene in neuronal cells activates astrocytes and produces vascular lesions that resemble this pathology 9 . this thus reinforces the importance of cellular crosstalk within the neurovascular unit . although nuclear accumulation of active b - catenin appears to be a significant characteristic of the mutated genotype , the inventors do not have direct indications of the processes that drive the b - catenin concentration into the nucleus of the ccm3 - knockout endothelial cells . in general , the issue of the molecular mechanisms that regulate nuclear accumulation of b - catenin remain virtually unknown ( for review , see 18 ). however , concomitant with the increase in concentration into the nucleus , the inventors observed that b - catenin dissociates from cell - cell junctions in both the inventors &# 39 ; in vitro and in vivo models of endothelial - cell - specific deletion of ccm3 . junctional b - catenin is mostly associated with ve - cadherin , the transmembrane constituent of the adherens junctions 43 , as well as with the b - catenin destruction complex 44 . in the ccm3 - knockout endothelial cells , the adherens junctions are disorganized , as also observed after ablation of both ccm1 45 , 46 and ccm2 6 . in addition , the b - catenin associated with ve - cadherin is reduced here , as has also been observed after ablation of ccm1 10 . speculate that this decreased association of b - catenin with ve - cadherin is accompanied by accumulation of active b - catenin in the nucleus . this concentration of active b - catenin into the nucleus characterizes conditions of decreased junction stability in endothelial cells , as observed in sparse endothelial cells and in ve - cadherin - knockout endothelial cells 17 . in both of these cases , the total amount of b - catenin is reduced , even to very low levels , although the residual active b - catenin accumulates in the nucleus . inhibition of proteosomal degradation with ‘ passive ’ redistribution of active b - catenin into the nucleus appears not to be likely , as the total amount of active b - catenin actually decreases . of particular interest , the inventors have identified two inhibitors of b - catenin transcription signaling , sulindac sulfide and sulindac sulfone , that also inhibit the development of vascular lesions in ccm3 - ecko mice . these agents are both nsaids that have significant chemopreventive efficacies against colon cancer in human patients , and they are under evaluation in experimental models of other types of cancer 47 - 52 . sulindac sulfone is potentially more interesting than the sulindac sulfide for therapy of pathologies characterized by vascular malformation such as ccm since it lacks anti - 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