Patent Application: US-201013138537-A

Abstract:
the invention is in the field of molecular medicine . it provides antagonistic compounds for frizzled 1 and / or frizzled - 2 receptors , which may be useful in molecular imaging of the wound healing process after myocardial infarction and in therapeutic intervention into wound healing after remodeling of the heart , thereby ameliorating the consequences of myocardial infarction . the invention provides a method for antagonizing frizzled - 1 or frizzled - 2 receptors , wherein the receptor is contacted with a composition comprising a linear fragment of wnt3 or wnt5a or a functional analogue thereof , which comprises at least one cysteine residue , one threonine residue , one aspartic acid residue and one glycine residue .

Description:
human embryonic kidney ( hek ) cells were used for screening . these cells have a luciferase construct stably transfected into the genome ( see fig2 a ), such that when the frizzled receptor is activated and β - catenin is increased , the luciferase is activated by transcription and light can be measured . the peptides were tested by transfecting the frizzled - 1 or the frizzled - 2 receptor and addition of um206 ( s - s ) to the cell culture . for testing antagonistic activity , um206 was added and the natural ligand wnt3a was added , which is a natural stimulus for the canonical pathway . fig3 shows the results for the antagonist um206 . for this assay , we used rat cardiac fibroblasts , which were immortalized with telomerase , the c - fit cell line . this cell line was characterized previously and resembles features of primary cardiac fibroblasts . c - fit cells were plated , treated when they were 70 % confluent and a wound was made with a pipette tip at the moment of 100 % confluence . previous research revealed that overexpression of fz2 with addition of wnt3a / wnt5a delayed the much needed migration of the c - fit cells into the wound . this assay was first tested with the natural ligand wnt3a , in combination with frizzled - 1 and - 2 overexpression . these results clearly indicated that wnt3a in combination with frizzled - 1 or - 2 inhibited the migration . next step was to see whether the antagonist could counteract this . ( see fig4 .) um206 inhibited the delaying effect of the natural ligands and the migration was reset to the migration speed of the control . for this assay , we used the same c - fit cells as for the migration assay to study a second component of wound healing , namely , differentiation of fibroblasts into myofibroblasts . when wound healing starts , cells called fibroblasts migrate into the scar . these cells have no contractile properties ; however , they can differentiate into myofibroblasts , which can actively contract . to study which signals influence the transition from fibroblast into myofibroblasts , we treated the c - fit cells and harvested them for mrna isolation . next , we tested them for the presence of specific markers for myofibroblasts by reverse transcription of the mrna and subsequent quantitative pcr analysis , which are absent in fibroblasts . one of those markers is α - smooth muscle actin , which makes the cells contract . the results are shown in fig6 . um206 was injected into the femoral artery of swiss mice ( concentration of 10 − 9 m ). blood was sampled at different time points in the femoral vein . the results are shown in fig7 . on the y - axis , the concentration of um206 in the plasma is shown . this is determined with hplc and the area under the peak is calculated and shown on the y - axis . all values were corrected for the baseline value . ( see fig7 .) analogues of um206 were prepared by solid phase peptide synthesis . each amino acid residue was sequentially replaced by an alanine residue and antagonistic activity of the analogue was tested as described in the activity assay of example 1 . fig8 shows the results for the alanine screening . titles indicate which amino acid residue was substituted by an alanine residue . essential amino acids for antagonistic activity of um206 were determined by an alanine scan ; furthermore , some additional modifications were tested . all peptides were tested at a concentration of 1 . 10 - 9 m . these results highlighted four important amino acids , namely : cysteine , threonine , aspartic acid and glycine . replacement of both cysteines by alanines or serines , or alkylation of both cysteines , abolished the antagonistic properties of um206 . simultaneous replacement of threonine , aspartic acid and glycine by alanine strongly reduced the antagonistic properties of um206 . the peptide cnvsshgidgcdl ( seq id no : 18 ), derived from the area of wnt3a homologous to um206 , showed antagonistic properties too , albeit that the potency was lower than that of um206 . all results are the average of three independent measurements and are represented as means +/− sem . (*** p & lt ; 0 . 001 .) linear peptides according to the invention may be forced into a cyclic structure at 10 to 100 μm under slightly alkaline conditions at ph 7 . 5 to 8 . 5 by aeration under slow bubbling , or stirring , at 5 ° c . to 25 ° c . for two hours to four days . oxidizing agents such as potassium ferricyanide or dimethyl sulfoxide ( dmso ) may also be added . an alternative way to create cyclic peptides is by creating a s — chr — s — bond , wherein r may be any group but preferably a hydrogen atom . such a bond may be created by reacting two sh groups with a formaldehyde equivalent , or with an aldehyde rch ═ o . reagent stock concentration volume single reaction master mix 2x 12 . 5 μl forward primer 15 pmol / μl 0 . 5 μl reverse primer 15 pmol / μl 0 . 5 μl mq 6 . 5 μl template 5 μl remove medium wash with 5 ml pbs remove pbs add 1 ml trypsine - edta add 9 ml medium put the 10 ml in 15 ml tube centrifuge for 5 minutes remove medium homogeniseren in 4 ml medium 1 ml in filterdop 25 cm 2 flask add 20 ml medium herein , the term “ medium ” describes medium with 10 % fcs and 1 % gentamycine . heat fugene6 and dna 15 minutes at rt 2 ml serum - free medium in tube add 3μl fugene add 2 μg dna incubate 30 minutes at rt wash with pbs add 50 μtransfectiemix add 1 ml antibiotica - free medium remove medium wash with pbs add 500 μl ⅕ lysisbuffer shake for 60 minutes centrifuge in tube 20 μl lysaat and 50 μl luciferase in new tube for measurement vortex measure 1 . spilt the cells 1 : 10 in 10 ml culture medium without antibiotics and let the cells grow for four days ( approximately to confluency ) 2 . take off the medium and sterile filter ( this is the first batch ) 3 . add 10 ml medium without antibiotics and let the cells grow for three days 4 . take off the medium and sterile filter ( this is the second batch ) stable at 4 ° c . and can be frozen . weigh the peptide weigh the resin make hbtu ratio : 7 . 6 g in 40 ml wash column with dmf activated 1 st peptide by addition of 2 ml hbtu and 0 . 5 ml ttc ( di - isopropyl ethylamine ) 1 minute activating 10 to 20 minutes peptide on column and stirring wash with dmf for 40 seconds ( flow wash ) two shots of taa ( trifluoroacetic acid ) shortly after each other and discard immediately 2 × 1 minute taa on kolom wash with dmsf wash with taa make : a = 5 ml dmsf with 0 . 5 m ( 236 microliter ) acetic anhydride b = 5 ml dmsf with 0 . 5 m ( 196 microliter ) pyridine 2 . 5 ml a and 2 . 5 ml b 2 minutes on kolom discard 2 . 5 ml a and 2 . 5 ml b 2 minutes on kolom discard flow wash dmsf wash with 250 ml 20 % piperdine in dmsf ( 8 minutes ) wash with dmf wash with dcm wash with dcm / etoh the alignment of wnt3a and 5a is shown in fig9 a . three areas of high homology that contained two cysteines are underlined ( um207 ), ( um208 ) and ( um206 ). in fig9 b through 9d , the inhibitory effect of these three peptides on wnt3a - induced luciferase activity in hek293 - supertopflash is shown . in each figure , the left set of columns shows that none of the three peptides by itself could induce an increase in luciferase activity in cells , whereas , addition of wnt3a increases luciferase activity ˜ 450 - fold . transfection of rfzd - 1 ( middle set of columns ) or rfzd - 2 ( right set of columns ) further increased the wnt3a - induced luciferase activity , an effect that could be blocked almost completely by administration of either um206 ( fig9 b ), um207 ( fig9 c ) or um208 ( fig9 d ) at a concentration of 1 . 10 − 8 m . a complete inhibition of wnt3a - induced topflash activation was also observed in cho and cos - 7 cells overexpressing either rfzd - 1 or - 2 , albeit that the fold induction of luciferase activity was considerably lower in these cells . again , the peptides by themselves had no effect on the luciferase activity . peptides derived from other regions showed no activity at concentrations up to 10 μm ( see table 3 )). to determine the inhibitory constant of um206 - 8 , hek293 - supertopflash cells were transfected with either rfzd - 1 or - 2 and incubated with wnt3a ( 10 − 9 m ) in the presence of increasing concentrations of either of the peptides ( fig1 ). for um206 , a biphasic inhibition curve was observed that was best fitted with a two - binding - state model . in hek293 - supertopflash cells transfected with rfzd - 2 , the high affinity site with an ic 50 of 1 . 69 ± 0 . 04 10 − 11 m was most prominent , whereas in rfzd - 1 transfected cells , the binding site with an ic50 of 2 . 1 ± 0 . 18 10 − 9 m was the major binding site . this suggests that um206 binds to rfzd - 2 with an approximate 100 - fold higher affinity than to rfzd - 1 . in contrast , the inhibition curves for um207 and um208 were best fitted with a single binding site model ; the ic 50 values for fzd - 2 were 1 . 52 ± 0 . 08 10 − 8 m and 2 . 11 ± 0 . 11 10 − 6 , and for fzd - 1 , 4 . 00 ± 0 . 07 10 − 8 m and 2 . 06 ± 0 . 09 10 − 6 m , respectively . these results clearly show that um206 has the highest affinity for rfzd - 1 and - 2 , so in the remainder of the experiments , this antagonist was used . in fig3 , panel e , the efficacy of um206 to block the wnt3a - induced activation of luciferase activity in hek293 supertopflash cells transfected with different fzds is shown . addition of um206 in a concentration of 1 . 10 − 8 m abolished the induction of luciferase activity almost completely in cells overexpressing rfzd - 1 and - 2 , but had no effect on cells overexpressing hfzd - 4 or - 5 . this was not due to species differences , because um206 was fully effective in antagonizing the activation of mouse and human fzd - 1 and - 2 by wnt3a as well ( data not shown ). from these results , we conclude that um206 is a high affinity antagonist for fzd - 1 and - 2 but that it does not block fzd - 4 and - 5 in a relevant concentration range . in table 2 , the effect of substitution of each of the individual amino acids by alanine on wnt3a - induced luciferase activity is shown . in this experiment , the antagonists were tested in a concentration of 1 . 10 − 9 m , allowing the detection of more subtle changes in antagonistic properties that could remain unnoticed at a concentration of 1 . 10 − 8 m . this ala - scan showed that replacement of threonine 4 , glycine 7 and aspartic acid 9 affected the inhibitory properties of um206 most strongly . the combination of these three replacements abolished the antagonizing properties almost completely . substitution of either of the cysteine residues at position 1 or 11 did not affect the antagonistic effect of um206 , but simultaneous substitution of both cysteine residues by either alanine or serine rendered um206 completely ineffective . substitution of the c - terminal glutamic acid and leucine by an alanine - alanine sequence reduced the inhibitory properties of um206 only slightly , whereas deletion of the c - terminal glutamic acid and leucine sequence led to a significant reduction of the potency of um206 . so , we conclude that the antagonistic activity of um206 is due to the proper spatial positioning of the three amino acid residues thr , gly and asp and to the presence of at least one cysteine residue . um206 antagonized effects of wnt3a and wnt5a on immortalized cardiac fibroblasts the overexpression of fzd - 1 and - 2 in migrating ( myo ) fibroblasts during infarct healing was one of the first reports of activation of wnt / fzd signaling in cardiovascular remodeling ( blankesteijn 1997 ). to assess the functional relevance of wnt / fzd signaling on cardiac fibroblast proliferation , migration and differentiation , a cardiac fibroblast cell line immortalized with telomerase was used , as previously described ( janhunen 2009 , laeremans 2009 ). as shown in fig1 , administration of either wnt3a or wnt5a attenuated the migration of cardiac fibroblasts immortalized with telomerase ( cfit ) overexpressing either rfzd - 1 or - 2 ( panels a and b , respectively ). the effects of wnt proteins on cell migration were blocked completely by addition of um206 ( 1 . 10 − 8 m ) to the culture medium . um206 reduced ventricular remodeling and prevented heart failure development after myocardial infarction in mice in order to determine the pharmacokinetic properties of um206 , we injected 15 ng of the compound into the tail vein of mice and monitored the concentration in the blood at different time points . the half - life turned out to be 83 . 5 ± 1 . 63 minutes ( n = 24 ; see fig1 ). this half - life , combined with the high affinity of um206 allowed us to test its effects on infarct healing in mice . to this end , we administered um206 subcutaneously by an osmotic minipump ( 6 microgram / kg / day ); the control group was equipped with a saline - filled minipump . the minipumps were implanted at the time of infarct induction and lasted for up to five weeks . um206 administration offered a dramatic improvement of survival : at five weeks after mi , 35 % of the saline - treated mice had died , whereas not a single mouse had died in the um206 - treated group ( fig1 , panel a ). post - mortem analysis revealed that the mortality in the saline - treated group was due to heart failure , as diagnosed by a wet , foamy aspect of the lungs and severely elevated wet lung weight . in the surviving saline - treated mice , wet lung weight was 40 % higher than in the um206 - treated mice at five weeks post - mi , suggesting development of heart failure in the surviving saline - treated mice as well ( fig1 , panel b ). echocardiography showed a doubling of the ejection fraction and a 40 % reduction in end diastolic volume of the left ventricle in the um206 - treated group , all indicative for a better preservation of cardiac function ( fig1 , panels c . 1 and c . 2 ). further details of the echocardiographic analysis are provided in table 4 . cardiac function was further assessed using a millar pressure recording catheter inserted into the left ventricle . the tangents on the pressure time curve were significantly steeper in the um206 - treated groups , resulting in a higher positive dp / dt and a lower negative dp / dt value ( fig1 , panels d . 1 and d . 2 ). additional hemodynamic parameters are presented in table 5 . these observations further underscore the beneficial effect of um206 treatment on cardiac function post - mi . macroscopic analysis of longitudinal sections of the infarcted hearts revealed that um206 treatment profoundly reduced infarct expansion and ventricular dilatation ( fig1 , panels a . 1 and a . 2 ) compared to saline treatment . this was confirmed by histological analysis of the infarct area . the infarct areas were significantly smaller in um206 - treated compared to saline - treated mice ( fig1 , panel b ), both at 2 and 5 weeks after mi . moreover , as shown in fig1 , panel c , the infarct areas were significantly thicker in the um206 - treated mice at both time points . myofibroblast numbers were more than four - fold higher in the um206 - treated mice at two weeks post - mi as compared to saline - treated mice . in both groups , we observed a similar decline in myofibroblast numbers between two and five weeks post - mi , resulting in a significantly elevated number at five weeks post - mi in the um206 - treated group ( fig1 , panel d ). the higher myofibroblast numbers in the um206 - treated group could be confirmed by significantly higher a - sma levels in the five - week - old um206 - treated infarcts , as determined by qpcr ( fig1 , panel e . 1 ) and western blotting ( fig1 , panel e . 2 ). these results suggest that an increased myofibroblast content contributes to the reduced infarct expansion and subsequent ventricular dilation in the um206 - treated groups . moreover , um206 treatment ( five weeks ) resulted in an increased amount of blood vessels ( fig1 , panel f ) and reduced collagen levels ( fig1 , panel g ) in the infarct area , compared to saline treatment . in our experiments , we did not observe any effects of um206 on other organs known to contain fzd - 2 , including kidney and small intestine . qpcr of several wnt target genes revealed no difference in the expression levels of these genes in animals treated with um206 for five weeks ( see tables 6 and 7 ). to demonstrate that the binding site of um206 on fzd - 1 and - 2 is shared with um207 and wnt3a , we developed rhodamine - labeled um206 . attachment of rhodamine to lys 3 of um206 did not affect its ic 50 value for blocking wnt3a - induced luciferase expression in hek293 supertopflash cells ( not shown ). as shown in fig1 , incubation of frozen sections of mouse kidney and gut with um206 - rhodamine ( 1 . 10 − 8 m ) for 15 minutes , followed by rinsing in ice - cold pbs , revealed staining of tubular epithelium and panneth cells as assessed by two - foton microscopy . specificity of the binding site was confirmed by prior incubation of the tissue sections with um207 ( 1 . 10 − 8 m ) or recombinant wnt3a ( 1 . 10 − 8 m ). both compounds completely abolished the binding of um206 - rhodamine to its receptors . these data strongly suggest that um206 and um207 utilize a binding site on fzd - 1 and - 2 that is also occupied by wnt3a . herein , we describe the design of antagonists for fzd - 1 and - 2 , based on areas of high homology between wnt3a and - 5a . based on a set of pre - defined criteria , we identified three peptidergic antagonists , which all could block the activation of fzd - 1 and - 2 by wnt3a and wnt5a ; we named the peptides um206 - 8 . because the ic 50 - value of um206 for fzd - 1 and - 2 was lower than that of um207 and um208 , we focused on um206 in the remainder of the studies . all three peptides feature two cysteines separated by four to ten amino acids . the gly , asp and thr residues are present in all three peptides and were shown to be important in the blocking effect of um206 . either of the two cysteines of um206 could be replaced by alanine with little consequences for the binding , but replacing both cysteines caused complete loss of activity . um206 blocked fzd - 1 - and - 2 - mediated wnt signaling effectively in hek293 supertopflash , cost and cho cells , as well as cardiac fibroblasts immortalized with telomerase . moreover , um206 administration had a beneficial effect on infarct healing by increasing myofibroblast and blood vessel numbers in the infarct area , preventing dilatation of the left ventricle , improving cardiac function and completely preventing heart failure . finally , by using rhodamine - labeled um206 , we could show that um206 and um207 occupy the same binding site , which is also used by wnt3a . because of the lack of pharmacological tools to intervene in wnt / fzd signaling in vivo , only genetic interventions have been reported so far in studies on the role of wnt signaling in infarct healing . barandon et al . were the first to show a beneficial effect of overexpression on infarct healing of frza , a bovine homologue of soluble frizzled - related protein - 1 ( sfrp1 ). they observed a decreased infarct size with elevated numbers of myofibroblasts and increased angiogenesis in the infarcted frza transgenic mice ( barandon et al ., 2003 ), which could be confirmed in the present study using an fzd - 1 and - 2 antagonist . interestingly , a reduction in infarct rupture frequency was also observed in the frza transgenic mice . in contrast , um206 treatment of 129s6 mice , a mouse strain with infarct rupture frequency of ˜ 60 % ( van den borne et al ., 2009 ), did not reduce the number of ruptured infarcts ( h . laeremans , unpublished observations ). this suggests that sfrp may not solely act by blocking of wnt signaling but by additional mechanisms as well . in the mean time , the beneficial effects of sfrp overexpression on infarct healing have been confirmed by kobayashi et al . ( kobayashi et al ., 2009 ). taken together , these reports support the observations of the present study and point to a beneficial effect of inhibiting wnt / fzd signaling on the wound - healing process after mi . we did not observe any adverse effects of chronic um206 administration , indicating that this therapeutic intervention is safe and does not interfere with normal physiological processes . one of the most likely molecular targets of um206 appears to reside in the myofibroblasts in the infarct area . the role of myofibroblasts in wound healing and tissue repair is well established . these cells are responsible for the contraction of skin wounds , thereby limiting the size of the scar that is formed at the site of injury ( hinz , 2007 ; hinz et al ., 2007 ). more recently , a similar role has been described for myofibroblasts in the infarct area ( ertl and frantz , 2005 ; frangogiannis , 2006 ; squires et al ., 2005 ; van den borne et al ., 2010 ), thereby preventing expansion of the infarct area ( hutchins and bulkley , 1978 ). in a study on the effect of genetic background on infarct healing in the mouse , we observed an inverse relationship between myofibroblast content and infarct dilatation ( van den borne et al ., 2009 ). moreover , myofibroblasts remain present in well - healed human infarcts for decades , but are scarce in dilated infarcts obtained from heart failure patients ( cleutjens et al ., 1999 ; willems et al ., 1994 ). previously , we have shown that myofibroblasts express fzd - 1 and - 2 during their migration into the infarct area ( blankesteijn et al ., 1997 ). overexpression of components of the wnt / fzd pathway affect the differentiation and migration of cardiac fibroblasts immortalized with telomerase ( h . laeremans et al ., manuscript submitted ). therefore , we conclude that the fzd - 1 and - 2 expressed on myofibroblasts during infarct healing play a functional role in the wound healing process after mi and can serve as a therapeutic target for intervention in this process . moreover , the results of the present study highlight the importance of targeting ( myo ) fibroblasts to preserve cardiac function in the remodeling heart ( brown et al ., 2005 ; sun et al ., 2002 ). um206 may well serve as a lead for the development of drugs that address this target . this is of particular importance because the drugs that are currently available to treat infarct healing post - mi have pleiotropic effects on inflammation , extracellular matrix deposition and cardiac remodeling ( jugdutt , 2008 ). in this study , we used rhodamine - labeled um206 to investigate the binding site of um206 and um207 on fzd - 1 and - 2 in kidney and small intestine . the binding of rhodamine - labeled um206 could be prevented by preincubation with either wnt3a or um207 . these experiments provide the pharmacological evidence that um206 and um207 use the same binding site on the fzd protein . moreover , this binding site is also used by wnt3a . these data suggest that um206 and um207 act as competitive antagonists for wnt3a . the biological activity of peptides derived from wnt5a has been shown previously by säfholm et al . ( säfholm et al ., 2005 ). in this study , an anti - migratory effect of these peptides on breast cancer cells was observed . although their peptide , named foxy5 , is identical to the c - terminal 6 amino acids of um206 , its site of action appears to be distinct from that of um206 for several reasons : 1 ) the activity of foxy5 was sensitive to a fzd - 5 blocking antibody , whereas um206 interacts with fzd - 1 and - 2 but not with fzd - 5 . 2 ) substitution of the only cysteine residue in foxy5 with alanine did not affect its binding , whereas in um206 , at least one cysteine has to be present for biological activity . 3 ) foxy5 actively inhibited breast cancer cell migration , thereby acting as a wnt5a mimetic , whereas um206 alone had no effect on cell migration and acts as a wnt3a / 5a antagonist . 4 ) foxy5 and related peptides are active at concentrations of 10 to 100 whereas the ic 50 for um206 is in the nanomolar range . in conclusion , the results of the present study clearly show that pharmacological targeting of fzd proteins can be a successful and safe approach to intervene in pathological processes such as myocardial infarction . in the mean time , the role of wnt / fzd signaling has been implicated in processes as diverse as stem cell differentiation ( reya and clevers , 2005 ; säfholm et al ., 2005 ), tumor metastasis ( lai et al ., 2009 ), bone metabolism ( piters et al ., 2008 ) and various neurological disorders ( de ferrari and moon , 2006 ). this calls for an extensive search for ligands for the different fzd proteins involved in these diseases . the hek supertopflash cell line was kindly provided by j . nathans and cho and cos - 7 cells were obtained from dsmz , braunschweig , germany . the cfit cell line was developed and characterized in our lab ( janhunen et al ., 2009 ). the topflash construct is from upstate ( millipore , billerica mass ., usa ). the peptides were synthesized in our lab , but large - scale synthesis of um206 was performed by chempep , miami fla ., usa . the cell lines were cultured in 75 cm 2 culture flasks ( corning , schiphol , the netherlands ) in dulbeco &# 39 ; s modified essential medium with l - glutamine ( 2 mm ), 10 % fetal calf serum ( invitrogen , merelbeke , belgium ), and 1 % gentamycin ( sigma - aldrich , zwijndrecht , the netherlands ). before starting the experiment , the cell lines were treated with plasmocin 25 μg / ml ( invivogen , toulose , france ) and tested with a mycoalert mycoplasma detection kit ( lonza , rockland me ., usa ). cells were transiently transfected with fugene6 ( roche , indianapolis ind ., usa ) with plasmid dna , pcdna3 . 1 / hygro ( invitrogen ) with frizzled - 1 / 2 / 4 or wnts 3a - 5a or with pcdna3 . 0 ( invitrogen ) containing the β - catenin or frizzled - 5 sequence . after transfection , the cells were cultured for 24 hours in conditioned medium , collected from the cultured l - cells , l - cells with wnt3a or 5a ( invitrogen ). at the same moment the antagonist was added . all peptide fragments were synthesized by manual solid - phase peptide synthesis on a 0 . 3 - 0 . 4 mmol scale using the in situ neutralization / activation procedure for boc -/ bzl - peptide synthesis as previously described ( schnolzer et al ., 1992 ), but using hctu instead of hbtu as a coupling reagent . mbha - polystyrene resin ( 1 meq / g ) was used as the solid support . the peptides were deprotected and cleaved from the resin by treatment with anhydrous hf for one hour at 0 ° c ., using 4 v -% p - cresol as a scavenger . following cleavage , the peptides were precipitated with ice - cold diethylether , dissolved in aqueous buffer containing 6 m gn . hcl , 0 . 1 m sodium acetate buffer ( ph 4 ) and purified by semi - preparative reversed - phase hplc . fractions containing the desired product were identified by esi - ms , pooled and lyophilized . for the luciferase experiments not in hek cells , the cells were additionally transfected with a topflash construct , eight tcf / lef binding sites cloned into the pta - luc vector . luciferase activity was measured using luciferase assay system ( promega , madison wis ., usa ). cells were plated on day 0 , and cultured until 70 % confluence before transfection or treatment . migration assays started 48 hours after transfection and / or treatment . the time point where the scratch was made , is indicated as 0 hours . scratch width was measured at this time point , and also after 6 , 12 , and 24 hours . rna was isolated using the trizol method ( invitrogen ). for the rt - pcr , the rna was transcribed to cdna with iscript ™ cdna synthesis kit ( bio - rad , hercules calif ., usa ), syber green ( eurogentec , ghent , belgium ) was used for the detection of cdna levels and cyclophilin served as the house keeping gene . in supplementary methods panel 5 , the primer sequences are shown . for western blot , cells were homogenized in ice - cold laemmli buffer and protein content was measured using the bca protein assay ( pierce biotechnology inc ., rockford ill ., usa ); proteins were denatured by boiling , separated on a 10 % sds - page gel , and transferred onto a hybond c nitrocellulose membrane ( amersham biosciences , little chalfont , united kingdom ). after blocking , membranes were incubated overnight at 4 ° c . with primary antibodies against β - catenin , α - sma ( both 1 : 2000 , bd biosciences , franklin lakes n . j ., usa ) or β - actin ( 1 : 2000 sigma - aldrich ). anti - mouse immunoglobulin g 1 / 5000 ( vector labs inc .) was used as the secondary antibody , and the membranes were developed using the supersignal west pico chemiluminescence kit ( pierce ). images of the blots were analyzed with image analysis software ( qwin leica , cambridge , united kingdom ). male swiss mice were used ( 10 - 12 weeks of age , charles river , maastricht , the netherlands ). the animals were randomly included into the three different experimental groups . for the pharmacokinetics , animals had a venous cannula where a bolus injection of um206 was given and blood samples were collected . in the treatment study , either um206 ( 6 μg / kg / day ) or saline were administrated by an osmotic minipump ( alzet 2002 or 2006 for two - and five - week treatments , respectively ; alzet , cupertino calif ., usa ). mi was induced , under isofluorane anesthesia as previously described ( van den borne et al ., 2009 ). all experimental procedures were approved by the committee for animal research of maastricht university . all animals were subjected to extensive echocardiography studies for the assessment of myocardial infarct size , lv cavity dimensions and ventricular function . echocardiography examination was performed under 2 % isofluorane . echocardiograms were recorded with philips sonos 5500 ultrasound system ( philips , eindhoven , the netherlands ) using a 20 - mhz linear probe . the animals were anaesthetized with urethane ( 2 . 5 m / g body weight , i . p ., sigma - aldrich ) followed by intubation and connected to a rodent ventilator ( hugo sachs , march - hugstetten , germany ). body temperature was maintained at 37 ° c . the mice were then allowed to stabilize prior to hemodynamic measurements . a high - fidelity catheter tip micromanometer ( mikro - tip1 . 4f , spr - 671 ; millar instruments , houston tx , usa ) was inserted through the right carotid artery into the left ventricular cavity . ventricular pressure was measured . maximal positive and negative pressure development (+ dp / dt and − dp / dt ) and heart rate were determined on a beat - to - beat basis . the heart was then stimulated by an i . v . ramp - infusion of dobutamine ( sigma - aldrich ) using a micro - injection pump ( model 200 series , kdscientific , boston , mass ., usa ). blood samples were collected from the animals for the pharmacokinetics and after 14 to 35 days treatment , to determine the concentration of um206 . blood was centrifuged and the plasma was treated with 1 % tfa . analytical hplc was performed using a vydac c18 rp - hplc column ( 4 . 6 min × 150 mm , 1 ml / minute flow rate ) connected to a varian prostar system consisting of two varian prostar 215 delivery modules and a varian prostar 320 uvnis detector ( 214 nm ). a linear gradient of 0 % to 67 % buffer b in buffer a over 30 minutes was used , where buffer a = 0 . 1 v -% tfa in h 2 o and buffer b = 0 . 1 v -% tfa , 10 v -% h 2 o in ch 3 cn . semi - preparative hplc was performed using vydac c18 rp - hplc columns ( 10 mm × 250 mm , 5 ml / minute flow rate or 22 mm × 250 mm , 10 ml / minute flow rate ) connected to a waters deltaprep system consisting of a waters prep lc controller and a waters 2487 dual wavelength absorbance detector ( 214 nm ). peptides were eluted using a shallow gradient of b in a , based on an exploratory analytical hplc run . product - containing fractions were analyzed by electrospray ionization mass spectrometry ( esi - ms ), pooled and lyophilized mass spectrometry . esi - ms was performed on an applied biosystems sciex api 150 ex electrospray ionization quadrupole ( esi - q ) mass spectrometer . peptide masses were calculated from the experimental mass to charge ( m / z ) ratios from all the protonation states observed in the esi - ms spectrum of a peptide using analyst 1 . 4 . 2 software ( sciex ). one - half of the hearts of the mice was embedded in paraffin and cut in 4 μm sections . the paraffin sections were rehydrated and washed in pbs . one section was stained with azan , allowing an accurate determination of the infarct size . to visualize the myofibroblasts in the infarct area , the sections were incubated with an antibody directed against a - smooth muscle actin ( sigma - aldrich ), followed by incubation with the peroxidase - conjugated secondary antibody ( vector laboratories ). nuclei were visualized by hematoxylin . photos were taken with a leica ( ctr500 , 63x / 0 . 85 ) camera and analyzed with the quantimet program ( qwin / qgo ). an examiner blinded to the groups of the animals obtained all measurements . for the experiments with rhodamine - labeled um206 , frozen sections ( 4 μm ) of mouse kidney and small intestine were used . all values are shown as mean ± s . e . m . differences between groups were examined for statistical significance using two - way anova with the bonferroni post - test or unpaired student t - test ( graph pad prism ). a p value less than 0 . 05 was considered as a statistically significant difference . blankesteijn w . m ., y . p . essers - janssen , m . m . ulrich , and j . f . smits , 1996 . increased expression of a homologue of drosophila tissue polarity gene “ frizzled ” in left ventricular hypertrophy in the rat , as identified by subtractive hybridization . j . mol . cell cardiol . 28 : 1187 - 91 . blankesteijn w . m ., y . p . essers - janssen , m . j . verluyten , m . j . daemen , and j . f . smits , 1997 . a homologue of drosophila tissue polarity gene frizzled is expressed in migrating myofibroblasts in the infarcted rat heart . nat med . 3 : 541 - 4 . brack a . s ., m . j . conboy , s . roy , m . lee , c . j . kuo , c . keller , and t . a . rando , 2007 . increased wnt signaling during aging alters muscle stem cell fate and increases fibrosis . science 317 : 807 - 10 . imai k ., m . morikawa , j . d &# 39 ; 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