Patent Application: US-43933703-A

Abstract:
a method for modulating vasculogenesis or arteriogenesis or angiogenesis , especially for treating heart and limb ischemia , using the core domain protein of pdgf - c , a new member of the pdgf / vegf family of growth factors , or a homodimer or a heterodimer comprising the core domain . also disclosed are pharmaceutical compositions comprising the core protein , nucleotide sequences encoding the protein , and uses thereof in medical and diagnostic applications .

Description:
[ 0154 ] fig1 ( seq id no : 2 ) shows the complete nucleotide sequence of cdna encoding a human pdgf - c ( hpdgf - c )( 2108 bp ), which is a new member of the vegf / pdgf family . a clone # 4 ( see fig3 and 4 — seq id nos : 4 and 5 ) encoding hpdgf - c was not full length and lacked approximately 80 base pairs of coding sequence when compared to the mouse protein ( corresponding to 27 amino acids ). additional cdna clones were isolated from a human fetal lung cdna library to obtain an insert which included this missing sequence . clone # 10 had a longer insert than clone # 4 . the insert of clone # 10 was sequenced in the 5 ′ region and it was found to contain the missing sequence . clone # 10 was found to include the full sequence of human pdgf - c . some 5 ′- untranslated sequence , the translated part of the cdna encoding human pdgf - c and some 3 ′- untranslated nucleotide sequence are shown in fig1 ( seq id no : 2 ). a stop codon in frame is located 21 bp upstream of the initiation atg ( the initiation atg is underlined in fig1 ). work to isolate this new human pdgf / vegf began after a search of the expressed sequence tag ( est ) database , dbest , at the national center for biotechnology information ( ncbi ) in washington , d . c ., identified a human est sequence ( w21436 ) which appears to encode part of the human homolog of the mouse pdgf - c . based on the human est sequence , two oligonucleotides were designed : 5 ′- gaa gtt gag gaa ccc agt g - 3 ′ ( seq id no : 25 ) forward 5 ′- ctt gcc aag aag ttg cca ag - 3 ′ ( seq id no : 26 ) reverse . these oligonucleotides were used to amplify by polymerase chain reaction ( pcr ) a polynucleotide of 348 bps from a human fetal lung 5 ′- stretch plus λgt10 cdna library , which was obtained commercially from clontech . the pcr product was cloned into the pcr 2 . 1 - vector of the original ta cloning kit ( invitrogen ). subsequently , the 348 bps cloned pcr product was used to construct a hpdgf - c probe according to standard techniques . 10 6 lambda - clones of the human fetal lung 5 ′- stretch plus λgt10 cdna library ( clontech ) were screened with the hpdgf - c probe according to standard procedures . among several positive clones , one , clone # 4 was analyzed more carefully and the nucleotide sequence of its insert was determined according to standard procedures using internal and vector oligonucleotides . the insert of clone # 4 contains a partial nucleotide sequence of the cdna encoding the full length human pdgf - c ( hpdgf - c ). the nucleotide sequence ( 1536 bp ) of the clone # 4 insert is shown in fig3 ( seq id no : 4 ). the translated portion of this cdna includes nucleotides 6 to 956 . the deduced amino acid sequence of the translated portion of the insert is illustrated in fig4 ( seq id no : 5 ). a polypeptide of this deduced amino acid sequence would lack the first 28 amino acid residues found in the full length hpdgf - c polypeptide . however , this polypeptide includes a proteolytic fragment which is sufficient to activate the pdgf alpha receptors . it should be noted that the first glycine ( gly ) of seq id no : 5 is not found in the full length hpdgf - c . a mouse est sequence ( ai020581 ) was identified in a database search of the dbest database at the ncbi in washington , d . c ., which appears to encode part of a new mouse pdgf , pdgf - c . large parts of the mouse cdna was obtained by pcr amplification using dna from a mouse embryo λgt10 cdna library as the template . to amplify the 3 ′ end of the cdna , a sense primer derived from the mouse est sequence was used ( the sequence of this primer was 5 ′- ctt cag tac ctt gga aga g , primer 1 ( seq id no : 27 )) to amplify the 5 ′ end of the cdna , an antisense primer derived from the mouse est was used ( the sequence of this primer was 5 ′- cgc ttg acc agg aga caa c , primer 2 ( seq id no : 28 )). the λgt10 vector primers were sense 5 ′- acg tga att cag caa gtt cag cct ggt taa ( primer 3 ( seq id no : 29 )) and antisense 5 ′- acg tgg atc ctg agt att tct tcc agg gta ( primer 4 ( seq id no : 30 )). combinations of the vector primers and the internal primers obtained from the mouse est were used in standard pcr reactions . the sizes of the amplified fragments were approx . 750 bp ( 3 ′- fragment ) and 800 bp ( 5 ′- fragment ), respectively . these fragments were cloned into the pcr 2 . 1 vector and subjected to nucleotide sequences analysis using vector primers and internal primers . since these fragments did not contain the full length sequence of mpdgf - c , a mouse liver zap cdna library was screened using standard conditions . a 261 bp 32 p - labeled pcr fragment was generated for use as a probe using primers 1 and 2 and using dna from the mouse embryo λgt10 library as the template ( see above ). several positive plaques were purified and the nucleotide sequence of the inserts were obtained following subcloning into pbluescript . vector specific primers and internal primers were used . by combining the nucleotide sequence information of the generated pcr clones and the isolated clone , the full length amino acid sequence of mpdgf - c could be deduced ( see fig6 )( seq id no : 7 ). [ 0159 ] fig7 shows a comparative sequence alignment of the mouse and human amino acid sequences of pdgf - c ( seq id nos : 6 and 2 , respectively ). the alignment shows that human and mouse pdgf - cs display an identity of about 87 % with 45 amino acid replacements found among the 345 residues of the full length proteins . almost all of the observed amino acid replacements are conservative in nature . the predicted cleavage site in mpdgf - c for the signal peptidase is between residues g19 and t20 . this would generate a secreted mouse peptide of 326 amino acid residues . [ 0160 ] fig8 provides a schematic domain structure of mouse pdgf - c with a signal sequence ( striped box ), a n - terminal cub domain and the c - terminal pdgf / vegf - homology domain ( open boxes ). the amino acid sequences denoted by the lines have no obvious similarities to cub domains or to vegf - homology domains . the high sequence identity suggests that human and mouse pdgf - c have an almost identical domain structure . amino acid sequence comparisons revealed that both mouse and human pdgf - c display a novel domain structure . apart from the pdgf / vegf - homology domain located in the c - terminal region in both proteins ( residues 164 to 345 ), the n - terminal region in both pdgf - cs have a domain referred to as a cub domain ( bork and beckmann , j . mol . biol ., 1993 231 : 539 - 545 ). this domain of about 110 amino acids ( amino acid residues 50 - 160 ) was originally identified in complement factors c1r / c1s , but has recently been identified in several other extracellular proteins including signaling molecules such as bone morphogenic protein 1 ( bmp - 1 ) ( wozney et al ., science , 1988 242 : 1528 - 1534 ) as well as in several receptor molecules such as neuropilin - 1 ( np - 1 ) ( soker et al ., cell , 1998 92 : 735 - 745 ). the functional roles of cub domains are not clear but it may participate in protein - protein interactions or in interactions with carbohydrates including heparin sulfate proteoglycans . [ 0162 ] fig9 shows the amino acid sequence alignment of the c - terminal pdgf / vegf - homology domains of human and mouse pdgf - cs with the c - terminal pdgf / vegf - homology domains of pdgf / vegf family members , vegf 165 , plgf - 2 , vegf - b 167 , pox orf vegf , vegf - c , vegf - d , pdgf - a and pdgf - b ( seq id nos : 8 - 17 ). some of the amino acid sequences in the n - and c - terminal regions in vegf - c and vegf - d have been deleted in this figure . gaps were introduced to optimize the alignment . this alignment was generated using the method of j . hein , ( methods enzymol . 1990 183 : 626 - 45 ) with pam250 residue weight table . the boxed residues indicate amino acids which match the pdgf - cs within two distance units . the alignment shows that pdgf - c has the expected pattern of invariant cysteine residues , a hallmark of members of this family , with one exception . between cysteine 3 and 4 , normally spaced by 2 residues there is an insertion of three extra amino acids ( nca ). this feature of the sequence in pdgf - c was highly unexpected . based on the amino acid sequence alignments in fig9 a phylogenetic tree was constructed and is shown in fig1 . the data show that the pdgf - c homology domain is closely related to the pdgf / vegf - homology domains of vegf - c and vegf - d . as shown in fig1 , the amino acid sequences from several cub - containing proteins were aligned ( seq id nos : 18 - 24 ). the results show that the single cub domain in human and mouse pdgf - c ( seq id nos : 18 and 19 , respectively ) displays a significant identify with the most closely related cub domains . sequences from human bmp - 1 , with 3 cub domains ( cub1 - 3 ( seq id nos : 20 - 22 )) and human neuropilin - 1 with 2 cub domains ( cub1 - 2 )( seq id nos : 23 and 24 , respectively ) are shown . gaps were introduced to optimize the alignment . this alignment was generated using the method of j . hein , ( methods enzymol ., 1990 183 626 - 45 ) with pam250 residue weight table . [ 0166 ] fig1 shows a northern blot analysis of the expression of pdgf - c transcripts in several human tissues . the analysis shows that pdgf - c is encoded by a major transcript of approximately 3 . 8 - 3 . 9 kb , and a minor of 2 . 8 kb . the numbers to the right refer to the size of the mrnas ( in kb ). the tissue expression of pdgf - c was determined by northern blotting using a commercial multiple tissue northern blot ( mtn , clontech ). the blots were hybridized at according to the instructions from the supplier using expresshyb solution at 68 ° c . for one hour ( high stringency conditions ), and probed with a 353 bp hpdgf - c est probe from the fetal lung cdna library screening as described above . the blots were subsequently washed at 50 ° c . in 2 × ssc with 0 . 05 % sds for 30 minutes and at 50 ° c . in 0 . 1 × ssc with 0 . 1 % sds for an additional 40 minutes . the blots were then put on film and exposed at − 70 ° c . the blots show that pdgf - c transcripts are most abundant in heart , liver , kidney , pancreas and ovary while lower levels of transcripts are present in most other tissues , including placenta , skeletal muscle and prostate . pdgf - c transcripts were below the level of detection in spleen , colon and peripheral blood leucocytes . [ 0167 ] fig1 shows the regulation of pdgf - c mrna expression by hypoxia . size markers ( in kb ) are indicated to the left in the lower panel . the estimated sizes of pdgf - c mrnas is indicated to the left in the upper panel ( 2 . 7 and 3 . 5 kbs , respectively ). to explore whether pdgf - c is induced by hypoxia , cultured human skin fibroblasts were exposed to hypoxia for 0 , 4 , 8 and 24 hours . poly ( a )+ mrna was isolated from cells using oligo - dt cellulose affinity purification . isolated mrnas were electrophoresed through 12 % agarose gels using 4 μg of mrna per line . a northern blot was made and hybridized with a probe for pdgf - c . the sizes of the two bands were determined by hybridizing the same filter with a mixture of hvegf , hvegf - b and hvegf - c probes ( enholm et al . oncogene , 1997 14 2475 - 2483 ), and interpolating on the basis of the known sizes of these mrnas . the results shown in fig1 indicate that pdgf - c is not regulated by hypoxia in human skin fibroblasts . [ 0168 ] fig1 shows the expression of pdgf - c mrna in human tumor cells lines . to explore whether pdgf - c was expressed in human tumor cell lines , poly ( a )+ mrna was isolated from several known tumor cell lines , the mrnas were electrophoresed through a 12 % agarose gel and analyzed by northern blotting and hybridization with the pdgf - c probe . the results shown in fig1 demonstrate that pdgf - c mrna is expressed in several types of human tumor cell lines such as jeg3 ( a human choriocarcinoma , atcc # htb - 36 ), g401 ( a wilms tumor , atcc # crl - 1441 ), dami ( a megakaryoblastic leukemia ), a549 ( a human lung carcinoma , atcc # ccl - 185 ) and hel ( a human erythroleukemia , atcc # tid - 180 ). it is contemplated that further growth of these pdgf - c expressing tumors can be inhibited by inhibiting pdgf - c , as well as using pdgf - c expression as a means of identifying specific types of tumors . two synthetic peptides were generated and then used to raise antibodies against human pdgf - c . the first synthetic peptide corresponds to residues 29 - 48 of the n - terminus of full length pdgf - c and includes an extra cysteine residue at the n - and c - terminus : ckfqfssnkeqngvqdpqherc ( seq id no : 31 ). the second synthetic peptide corresponds to residues 230 - 250 of the internal region of full length pdgf - c and includes an extra cysteine residue at the c - terminus : grksrvvdlnllteevrlysc ( seq id no : 32 ). the two peptides were each conjugated to the carrier protein keyhole limpet hemocyanin ( klh , calbiochem ) using n - succinimidyl 3 -( 2 - pyridyldithio ) propionate ( spdp ) ( pharmacia inc .) according to the instructions of the supplier . 200 - 300 micrograms of the conjugates in phosphate buffered saline ( pbs ) were separately emulsified in freunds complete adjuvant and injected subcutaneously at multiple sites in rabbits . the rabbits were boostered subcutaneously at biweekly intervals with the same amount of the conjugates emulsified in freunds incomplete adjuvant . blood was drawn and collected from the rabbits . the sera were prepared using standard procedures known to those skilled in the art . the full length cdna encoding human pdgf - c was cloned into the mammalian expression vector , psg5 ( stratagene , la jolla , calif .) that has the sv40 promoter . cos - 1 cells were transfected with this construct and in separate transfections , with a psg5 vector without the cdna insert for a control , using the deae - dextran procedure . serum free medium was added to the transfected cos - 1 cells 24 hours after the transfections and aliquots containing the secreted proteins were collected for a 24 hour period after the addition of the medium . these aliquots were subjected to precipitation using ice cold 10 % trichloroacetic acid for 30 minutes , and the precipitates were washed with acetone . the precipitated proteins were dissolved in sds loading buffer under reducing conditions and separated on a sds - page gel using standard procedures . the separated proteins were electrotransferred onto hybond filter and immunoblotted using a rabbit antiserum against the internal peptide of full length pdgf - c , the preparation of which is described above . bound antibodies were detected using enhanced chemiluminescence ( ecl , amersham inc .). fig1 shows the results of this immunoblot . the sample was only partially reduced and the monomer of the human pdgf - c migrated as a 55 kda species ( the lower band ) and the dimer migrated as a 100 kda species ( upper band ). this indicates that the protein is secreted intact and that no major proteolytic processing occurs during secretion of the molecule in mammalian cells . example 3 : expression of full length and truncated human pdgf - c in baculovirus infected sf9 cells . the full length coding part of the human pdgf - c cdna ( 970 bp ) was amplified by pcr using deep vent dna polymerase ( biolabs ) using standard conditions and procedures . the full length pdgf - c was amplified for 30 cycles , where each cycle consisted of one minute denaturization at 94 ° c ., one minute annealing at 56 ° c . and two minutes extension at 72 ° c . the forward primer used was 5 ′ cgggatcccgaatccaacctgagtag3 ′ ( seq id no : 33 ). this primer includes a bamhi site ( underlined ) for in frame cloning . the reverse primer used was : this primer includes an ecori site ( underlined ) and sequences coding for a c - terminal 6 × his tag preceded by an enterokinase site . in addition , residues 230 - 345 of the pdgf / vegf homology domain ( pvhd ) i . e . the core domain protein of human pdgf - c were amplified by pcr using deep vent dna polymerase ( biolabs ) using standard conditions and procedures . the residues 230 - 345 of the pvhd of pdgf - c were amplified for 25 cycles , where each cycle consisted of one minute denaturization at 94 ° c ., four minutes annealing at 56 ° c . and four minutes extension at 72 ° c . the forward primer used was 5 ′ cggatcccggaagaaaatcca gagtggtg3 ′ ( seq id no : 35 ). this primer includes a bamhi site ( underlined ) for in frame cloning . the reverse primer used was this primer includes an ecori site ( underlined ) and sequences coding for a c - terminal 6 × his tag preceded by an enterokinase site . the pcr products were digested with bamhi and ecori and subsequently cloned into the baculovirus expression vector , pacgp67a . verification of the correct sequence of the pcr products cloned into the constructs was by nucleotide sequencing . the expression vectors were then co - transfected with baculogold linearized baculovirus dna into sf9 insect cells according to the manufactures protocol ( pharmingen ). recombined baculovirus were amplified several times before beginning large scale protein production and protein purification according to the manual ( pharmingen ). sf9 cells , adapted to serum free medium , were infected with recombinant baculovirus at a multiplicity of infection of about 7 . media containing the recombinant proteins were harvested 4 days after infection and were incubated with ni - nta - agarose beads ( qiagen ). the beads were collected in a column and after extensive washing with 50 mm sodium phosphate buffer ph 8 , containing 300 mm nacl ( the washing buffer ), the bound proteins were eluted with increasing concentrations of imidazole ( from 100 mm to 500 mm ) in the washing buffer . the eluted proteins were analyzed by sds - page using 12 . 5 % polyacrylamide gels under reducing and non - reducing conditions . for immunoblotting analyses , the proteins were electrotransferred onto hybond filters for 45 minutes . fig1 a - c show the isolation and partial characterization of full length human pdgf - c protein . in fig1 a , the recombinant full length protein was visualized on the blot using antipeptide antibodies against the n - terminal peptide ( described above ). in fig1 b , the recombinant full length protein was visualized on the blot using antipeptide antibodies against the internal peptide ( described above ). the separated proteins were visualized by staining with coomassie brilliant blue ( fig1 c ). the numbers at the bottom of fig1 a - c refer to the concentration of imidazole used to elute the protein from the ni - nta column and are expressed in molarity ( m ). fig1 a - c also show that the full length protein migrates as a 90 kda species under non - reducing conditions and as a 55 kda species under reducing conditions . this indicates that the full length protein was expressed as a disulfide - linked dimer . fig1 a - c show the analysis of the isolation and partial characterization of a truncated form of human pdgf - c containing the pdgf / vegf homology domain only . in fig1 a , the immunoblot analysis of fractions eluted from the ni - agarose column demonstrates that the protein could be eluted at imidazole concentrations ranging between 100 - 500 mm . the eluted fractions were analyzed under non - reducing conditions , and the truncated human pdgf - c was visualized on the blot using antipeptide antibodies against the internal peptide ( described above ). fig1 b shows the coomassie brilliant blue staining of the same fractions as in fig1 a . this shows that the procedure generates highly purified material migrating as a 36 kda species . fig1 c shows the coomassie brilliant blue staining of non - reduced ( non - red .) and reduced ( red .) truncated human pdgf - c protein . the data show that the protein is a secreted dimer held together by disulfide bonds and that the monomer migrates as a 24 kda species . to assess the interactions between full length and truncated pdgf - c and the vegf receptors , full length and truncated pdgf - c were tested for their capacity to bind to soluble ig - fusion proteins containing the extracellular domains of human vegfr - 1 , vegfr - 2 and vegfr - 3 ( olofsson et al ., proc . natl . acad . sci . usa , 1998 95 : 11709 - 11714 ). the fusion proteins , designated vegfr - 1 - ig , vegfr - 2 - ig and vegfr - 3 - ig , were transiently expressed in human 293 ebna cells . all ig fusion proteins were human vegfrs . cells were incubated for 24 hours after transfection , washed with dulbecco &# 39 ; s modified eagle medium ( dmem ) containing 0 . 2 % bovine serum albumin and starved for 24 hours . the fusion proteins were then precipitated from the clarified conditioned medium using protein a - sepharose beads ( pharmacia ). the beads were combined with 100 microliters of 10 × binding buffer ( 5 % bovine serum albumin , 0 . 2 % tween 20 and 10 μg / ml heparin ) and 900 microliter of conditioned medium from 293 cells that had been transfected with mammalian expression plasmids encoding full length or truncated pdgf - c or control vector , then metabolically labeled with 35 s - cysteine and methionine ( promix , amersham ) for 4 to 6 hours . after 2 . 5 hours , at room temperature , the sepharose beads were washed 3 times with binding buffer at 4 ° c ., once with phosphate buffered saline and boiled in sds - page buffer . labeled proteins that were bound to the ig - fusion proteins were analyzed by sds - page under reducing conditions . radiolabeled proteins were detected using a phosphorimager analyzer . in all these analyses , radiolabeled pdgf - c failed to show any interaction with any of the vegf receptors . next , full length and truncated pdgf - c were tested for their capacity to bind to human pdgf receptors alpha and beta by analyzing their abilities to compete with pdgf - bb for binding to pdgf receptors . the binding experiments were performed on porcine aortic endothelial - 1 ( pae - 1 ) cells stably expressing the human pdgf receptors alpha and beta ( eriksson et al ., embo j , 1992 , 11 , 543 - 550 ). binding experiments were performed essentially as in heldin et al . ( embo j , 1988 , 7 : 1387 - 1393 ). different concentrations of human full - length and truncated pdgf - c , or human pdgf - bb were mixed with 5 ng / ml of 125 i - pdgf - bb in binding buffer ( pbs containing 1 mg / ml of bovine serum albumin ). aliquots were incubated with the receptor expressing pae - 1 cells plated in 24 - well culture dishes on ice for 90 minutes . after three washes with binding buffer , cell - bound 125 i - pdgf - bb was extracted by lysis of cells in 20 mm tris - hcl , ph 7 . 5 , 10 % glycerol , 1 % triton x - 100 . the amount of cell bound radioactivity was determined in a gamma - counter . a standard curve for the binding of 125 i - labeled pdgf bb homodimers to pae - 1 cells expressing pdgf alpha - receptor is shown in fig1 . an increasing excess of the unlabeled protein added to the incubations competed efficiently with cell association of the radiolabeled tracer . [ 0180 ] fig1 graphically shows that the truncated pdgf - c efficiently competed for binding to the pdgf alpha - receptor , while the full length protein did not . both the full length and truncated proteins failed to compete for binding to the pdgf beta - receptor . to test if pdgf - c causes increased phosphorylation of the pdgf alpha - receptor , full length and truncated pdgf - c were tested for their capacity to bind to the pdgf alpha - receptor and stimulate increased phosphorylation . serum - starved porcine aortic endothelial ( pae ) cells stably expressing the human pdgf alpha - receptor were incubated on ice for 90 minutes with pbs supplemented with 1 mg / ml bsa and 10 ng / ml of pdgf - aa , 100 ng / ml of full length human pdgf - cc homodimers ( flpdgf - cc ), 100 ng / ml of truncated pdgf - cc homodimers ( cpdgf - cc ), or a mixture of 10 ng / ml of pdgf - aa and 100 ng / ml of truncated pdgf - cc . full length and truncated pdgf - cc homodimers were produced as described above . sixty minutes after the addition of the polypeptides , the cells were lysed in lysis buffer ( 20 mm tris - hcl , ph 7 . 5 , 0 . 5 % triton x - 100 , 0 . 5 % deoxycholic acid , 10 mm edta , 1 mm orthovanadate , 1 mm pmsf 1 % trasylol ). the pdgf alpha - receptors were immunoprecipitated from cleared lysates with rabbit antisera against the human pdgf alpha - receptor ( eriksson et al ., embo j , 1992 11 : 543 - 550 ). the precipitated receptors were applied to a sds - page gel . after sds gel electrophoresis , the precipitated receptors were transferred to nitrocellulose filters , and the filters were probed with anti - phosphotyrosine antibody py - 20 , ( transduction laboratories ). the filters were then incubated with horseradish peroxidase - conjugated anti - mouse antibodies . bound antibodies were detected using enhanced chemiluminescence ( ecl , amersham inc ). the filters were then stripped and reprobed with the pdgf alpha - receptor rabbit antisera , and the amount of receptors was determined by incubation with horseradish peroxidase - conjugated anti - rabbit antibodies . bound antibodies were detected using enhanced chemiluminescence ( ecl , amersham inc ). the probing of the filters with pdgf alpha - receptor antibodies confirmed that equal amounts of the receptor were present in all lanes . pdgf - aa is included in the experiment as a control . fig2 shows that truncated , but not full length pdgf - cc , efficiently induced pdgf alpha - receptor tyrosine phosphorylation . this indicates that truncated pdgf - cc is a potent pdgf alpha - receptor agonist . [ 0182 ] fig2 shows the mitogenic activities of truncated and full length pdgf - cc on fibroblasts . the assay was performed essentially as described in mori et al ., j . biol . chem ., 1991 266 : 21158 - 21164 . serum starved human foreskin fibroblasts were incubated for 24 hours with 1 ml of serum - free medium supplemented with 1 mg / ml bsa and 3 ng / ml , 10 ng / ml or 30 ng / ml of full length pdgf - cc ( flpdgf - cc ), truncated pdgf - cc ( cpdgf - cc ) or pdgf - aa in the presence of 0 . 2 μmci [ 3h ] thymidine . after trichloroacetic acid ( tca ) precipitation , the incorporation of [ 3h ] thymidine into dna was determined using a beta - counter . the results show that truncated pdgf - cc , but not full length pdgf - cc , is a potent mitogen for fibroblasts . pdgf - aa is included in the experiment as a control . pdgf - c does not bind to any of the known vegf receptors . pdgf - c is the only vegf family member , thus far , which can bind to and increase phosphorylation of the pdgf alpha - receptor . pdgf - c is also the only vegf family member , thus far , to be a potent mitogen of fibroblasts . these characteristics indicate that the truncated form of pdgf - c may not be a vegf family member , but instead a novel pdgf . furthermore , the full length protein is likely to be a latent growth factor that needs to be activated by proteolytic processing to release the active pdgf / vegf homology domain . a putative proteolytic site is the dibasic motif found in residues 231 - 234 in the full length protein , residues - r - k - s - r -. this site is structurally conserved in a comparison between mouse and human pdgf - cs ( fig7 ). preferred proteases include , but are not limited to , factor x and enterokinase . the n - terminal cub domain may be expressed as an inhibitory domain which might be used to localize this latent growth factor in some extracellular compartment ( for example the extracellular matrix ) and which is removed by limited proteolysis when need , for example during embryonic development , tissue regeneration , tissue remodelling including bone remodelling , active angiogenesis , tumor progression , tumor invasion , metastasis formation and / or wound healing . to assess the interactions between truncated pdgf - c and the pdgf alpha and beta receptors , truncated pdgf - c was tested for its capacity to bind to porcine aortic endothelial - 1 ( pae - 1 ) cells expressing pdgf alpha or beta receptors , respectively ( eriksson et al ., embo j , 1992 11 : 543 - 550 ). the binding experiments were performed essentially as described in heldin et al . ( embo j , 1988 7 : 1387 - 1393 ). five micrograms of truncated pdgf - c protein in ten microliters of sodium borate buffer was radiolabeled using the bolton - hunter reagent ( amersham ) to a specific activity of 4 × 10 5 cpm / ng . different concentrations of radiolabeled truncated pdgf - c , with or without added unlabeled protein , in binding buffer ( pbs containing 1 mg / ml of bovine serum albumin ) was added to the receptor expressing pae - 1 cells plated in 24 - well culture dishes on ice for 90 minutes . after three washes with binding buffer , cell - bound 125 i - labeled pdgf - c was extracted by lysis of cells in 20 mm tris - hcl , ph 7 . 5 , 10 % glycerol , 1 % triton x - 100 . the amount of cell - bound radioactivity was determined in a gamma - counter . non - specific binding was estimated by including a 100 - fold molar excess of truncated pdgf - c in some experiments . all binding data represents the mean of triplicate analyses and the experimental variation in the experiment varied between 10 - 15 %. as seen in fig2 , truncated pdgf - c binds to cells expressing pdgf alpha receptors , but not to beta receptor expressing cells . the binding was specific as radiolabeled pdgf - c was quantitatively displaced by a 100 - fold molar excess of unlabeled protein . to demonstrate that full length pdgf - c can be activated by limited proteolysis to release the pdgf / vegf homology domain from the cub domain , the full length protein was digested with different proteases . for example , full length pdgf - c was digested with plasmin in 20 mm tris - hcl ( ph 7 . 5 ) containing 1 mm cacl 2 , 1 mm mgcl2 and 0 . 01 % tween 20 for 1 . 5 to 4 . 5 hours at 37 ° c . using two to three units of plasmin ( sigma ) per ml . the released domain essentially corresponded in size to the truncated pdgf - c species previously produced in insect cells . plasmin - digested pdgf - c and undigested full length pdgf - c were applied to a sds - page gel under reducing conditions . after sds - page gel electrophoresis , the respective proteins were transferred to a nitrocellulose filter , and the filter was probed using a rabbit antipeptide antiserum to residues 230 - 250 in full length protein ( residues grksrvvdlnllteevrlysc ( seq id no : 37 ) located in just n - terminal to the pdgf / vegf homology domain ). bound antibodies were detected using enhanced chemiluminescence ( ecl , amersham inc ). fig2 shows the immunoblot with a 55 kda undigested full length protein and the plasmin - generated 26 - 28 kda species . to assess the interactions between plasmin - digested pdgf - c and the pdgf alpha receptors , plasmin - digested pdgf - c was tested for its capacity to bind to porcine aortic endothelial - 1 ( pae - 1 ) cells expressing pdgf alpha receptors ( eriksson et al ., embo j , 1992 11 : 543 - 550 ). the receptor binding analyses were performed essentially as in example 7 using 30 ng / ml of 125 i - labeled truncated pdgf - c as the tracer . as seen in fig2 , increasing concentrations of plasmin - digested pdgf - c efficiently competed for binding to the pdgf alpha receptors . in contrast , undigested full length pdgf - c failed to compete for receptor binding . these data indicate that full length pdgf - c is a latent growth factor unable to interact with pdgf alpha receptors and that limited proteolysis , which releases the c - terminal pdgf / vegf homology domain , is necessary to generate an active pdgf alpha receptor ligand / agonist . a human pdgf - c 430 bp cdna fragment encoding the cub domain ( amino acid residues 23 - 159 in full length pdgf - c ) was amplified by pcr using deep vent dna polymerase ( biolabs ) using standard conditions and procedures . the forward primer used was this primer includes a bamhi site ( underlined ) for in clone frame cloning . the reverse primer used was this primer includes an ecori site ( underlined ) and sequences coding for a c - terminal 6 × his tag preceded by an enterokinase site . the amplified pcr fragment was subsequently cloned into a pacgp67a transfer vector . verification of the correct sequence of the expression construct , cub - pacgp67a , was by automatic nucleotide sequencing . the expression vectors were then co - transfected with baculogold linearized baculovirus dna into sf9 insect cells according to the manufacture &# 39 ; s protocol ( pharmingen ). recombined baculovirus were amplified several times before beginning large scale protein production and protein purification according to the manual ( pharmingen ). sf9 cells , adapted to serum free medium , were infected with recombinant baculovirus at a multiplicity of infection of about 7 . media containing the recombinant proteins were harvested 72 hours after infection and were incubated with ni - nta - agarose beads ( qiagen ) overnight at 4 ° c . the beads were collected in a column and after extensive washing with 50 mm sodium phosphate buffer ph 8 , containing 300 mm nacl ( the washing buffer ), the bound proteins were eluted with increasing concentrations of imidazole ( from 100 mm to 400 mm ) in the washing buffer . the eluted proteins were analyzed by sds - page using a polyacrylamide gel under reducing and non - reducing conditions . [ 0192 ] fig2 shows the results from coomassie blue staining of the gel . the human pdgf - c cub domain is a disulfide - linked homodimer with a molecular weight of about 55 kd under non - reducing conditions , while two monomers of about 25 and 30 kd respectively are present under reducing conditions . the heterogeneity is probably due to heterogenous glycosylation of the two putative n - linked glycosylation sites present in the cub domain at amino acid positions 25 and 55 . a protein marker lane is shown to the left in the figure . to gain insight into the biological function of pdgf - c , pdgf - c expression in mouse embryos was localized by non - radioactive in situ hybridization in tissue sections from the head ( fig2 a - 26 s ) and urogenital tract ( fig2 t - 26 v ) regions . the non - radioactive in situ hybridization employed protocols and pdgf - a and pdgfr - alpha probes are described in boström et al ., cell , 1996 85 : 863 - 873 , which is hereby incorporated by reference . the pdgf - c probe was derived from a mouse pdgf - c cdna . the hybridization patterns shown in fig2 a - 26 v are for embryos aged e16 . 5 , but analogous patterns are seen at e14 . 5 , e15 . 5 and e17 . 5 . sense probes were used as controls and gave no consistent pattern of hybridization to the sections . [ 0194 ] fig2 a shows the frontal section through the mouth cavity at the level of the tooth anlagen ( t ). the arrows point to sites of pdgf - c expression in the oral ectoderm . also shown is the tongue ( to ). fig2 b - 26 d show pdgf - c expression in epithelial cells of the developing tooth canal . individual cells are strongly labeled in this area ( arrow in fig2 d ), as well as in the developing palate ectoderm ( right arrow in fig2 c ). fig2 e shows the frontal section through the eye , where pdgf - c expression is seen in the hair follicles ( double arrow ) and in the developing eyelid . also shown is the retina ( r ). in fig2 f and 26g , the pdgf - c expression is found in the outer root sheath of the developing hair follicle epithelium . in fig2 h , pdgf - c expression is shown in the developing eyelid . there is an occurrence of individual strongly pdgf - c positive cells in the developing opening . also shown is the lens ( l ). in fig2 , pdgf - c expression in the developing lacrimal gland is shown by the arrow . in fig2 j , pdgf - c expression in the developing external ear is shown . expression is seen in the external auditory meatus ( left arrow ) and in the epidermal cleft separating the prospective auricle ( e ). fig2 k and 26l show pdgf - c expression in the cochlea . expression is seen in the semi - circular canals ( arrows in 26k ). there is a polarized distribution of pdgf - c mrna in epithelial cells adjacent to the developing hair cells ( arrow in 26l ). fig2 m and 26n show pdgf - c expression in the oral cavity . horizontal sections show expression in buccal epithelium ( arrows in 26m ) and in the forming cleft between the lower lip buccal and the gingival epithelium ( arrows in 26n ). also shown is the tooth anlagen ( t ) and the tongue ( to ). fig2 o and 26p show pdgf - c expression in the developing nostrils , shown on horizontal sections . pdgf - c expression appears strongest before stratification of the epithelium and the formation of the canal proper ( arrows in 260 and 26p ). also shown is the developing nostrils ( n ). fig2 q - 26 s show pdgf - c expression in developing salivary glands and ducts . fig2 q is the sublingual gland . fig2 r and 26s show the maxillary glands , the salivary gland ( sg ) and the salivary duct ( sd ). fig2 t - 26 v show the expression of pdgf - c in the urogenital tract . fig2 t shows the expression of pdgf - c in the developing kidney metanephric mesoderm . fig2 u shows the expression of pdgf - c in the urethra ( ua ) and in epithelium surrounding the developing penis . fig2 v shows the pdgf - c expression in the developing ureter ( u ). one of the strongest sites of pdgf - c expression is the developing kidney and so expression of pdgf - c , pdgf - a and pdgfr - alpha was looked at in the developing kidney . fig2 a - 27 f show the results of non - radioactive in situ hybridization demonstrating the expression ( blue staining in unstained background visualized using dic optics ) of mrna for pdgf - c ( fig2 a and 27b ), pdgf - a ( fig2 c and 27d ) and pdgfr - alpha ( fig2 e and 27f ) in e16 . 5 kidneys . the white hatched line in fig2 b , 27d and 27 f outlines the cortex border . the bar in fig2 a , 27c and 27 e represents 250 μm , and in fig2 b , 27d and 27 f represents 50 μm . pdgf - c expression is seen in the metanephric mesenchyme ( mm in fig2 a ), and appears to be upregulated in the condensed mesenchyme ( arrows in fig2 b ) undergoing epithelial conversion as a prelude to tubular development , which is situated on each side of the ureter bud ( ub ). pdgf - c expression remains at lower levels in the early nephronal epithelial aggregates ( arrowheads in b ), but is absent from mature glomeruli ( gl ) and tubular structures . pdgf - a expression is not seen in these early aggregates , but is strong in later stages of tubular development ( fig2 c and 24d ). pdgf - a is expressed in early nephronal epithelial aggregates ( arrowheads in fig2 d ), but once the nephron is developed further , pdgf - a expression becomes restricted to the developing henle &# 39 ; s loop ( arrow in fig2 d ). the strongest expression is seen in the henle &# 39 ; s loops in the developing marrow ( arrows in fig2 c ). the branching ureter ( u ) and the ureter bud ( ub ) is negative for pdgf - a . thus , the pdgf - c and pdgf - a expression patterns in the developing nephron are spatially and temporally distinct . pdgf - c is expressed in the earliest stages ( mesenchymal aggregates ) and pdgf - a in the latest stages ( henle &# 39 ; s loop formation ) of nephron development . pdgfr - alpha is expressed throughout the mesenchyme of the developing kidney ( fig2 e and 27f ) and may hence be targeted by both pdgf - c and pdgf - a . pdgf - b expression is also seen in the developing kidney , but occurs only in vascular endothelial cells . pdgfr - beta expression takes place in perivascular mesenchyme , and its activation by pdgf - b is critical for mesangial cell recruitment into glomeruli . these results demonstrate that pdgf - c expression occurs in close spatial relationship to sites of pdgfr - alpha expression , and are distinct from the expression sites of pdgf - a or pdgf - b . this indicates that pdgf - c may act through pdgfr - alpha in vivo , and may have functions that are not shared with pdgf - a and pdgf - b . since the unique expression pattern of pdgf - c in the developing kidney indicates a function as a pdgfr - alpha agonist separate from that of pdgf - a or - b , a comparison was made to the histology of embryonic day 16 . 5 kidneys from pdgfr - alpha knockout mice ( fig2 b and 28f ) with kidneys from wildtype ( fig2 a and 28c ), pdgf - a knockout ( fig2 d ) and pdgf - a / pdgf - b double knockout ( fig2 e ) mice . the bar in fig2 a and 28b represents 250 μm , and in fig2 c - 28 f represents 50 μm . heterozygote mutants of pdgf - a , pdgf - b and pdgfr - alpha ( boström et al ., cell , 1996 85 : 863 - 873 ; levéen et al ., genes dev ., 1994 8 : 18751887 ; soriano et al ., development , 1997 124 : 2691 - 70 ) were bred as c57b16 / 129sv hybrids and intercrossed to produce homozygous mutant embryos . pdgf - a / pdgf - b heterozygote mutants were crossed to generate double pdgf - a / pdgf - b knockout embryos . due to a high degree of lethality of pdgf - a −/− embryos before e10 ( boström et al ., cell , 1996 85 : 863 - 873 ), the proportion of double knockout e16 . 5 embryos obtained in such crosses were less than 1 / 40 . the histology of kidney phenotypes was verified on at least two embryos of each genotype , except the pdgf - a / pdgf - b double knockout for which only a single embryo was obtained . it is interesting that there is lack of interstitial mesenchyme in the cortex of pdgfr - alpha −/− kidney ( arrows in fig2 a and asterisk in fig2 f ) and the presence of interstitial mesenchyme in all other genotypes ( asterisks in fig2 c - e ). the branching ureter ( u ) and the metanephric mesenchyme ( mm ) and its epithelial derivatives appear normal in all mutants . the abnormal glomerulus in the pdgf - a / pdgf - b double knockout reflect failure of mesangial cell recruitment into the glomerular tuft due to the absence of pdgf - b . these results indicate that pdgfr - alpha knockouts have a kidney phenotype , which is not seen in pdgf - a or pdgf - a / pdgf - b knockouts , hence potentially reflecting loss of signaling by pdgf - c . the phenotype consists of the marked loss of interstitial mesenchyme in the developing kidney cortex . the cells lost in pdgfr - alpha −/− kidneys are thus normally pdgfr - alpha positive cells adjacent to the site of expression of pdgf - c . endogenous pdgf - c from human fibroblastic ag1523 cells is expressed as two principal species of about m r 25k , corresponding to processed pdgf - c , and a minor species of m r 55k , corresponding to the full - length protein . to obtain further information on the proteolytic process , serum - free medium was collected from ˜ 80 % confluent ag1523 cells . tca - precipitated proteins from 1 ml of medium were subjected to sds - page using a 12 % polyacrylamide gel ( biorad ) under reducing conditions and then immunoblotted . endogenous pdgf - c was detected using a rabbit anti - peptide antiserum against an internal peptide located in the human pdgf - cc core domain ( li et al ., 2000 ). bound antibodies were observed using enhanced chemiluminiscence plus ( ecl +; amersham ). as seen in fig2 , two principal m r 25 kda species can be seen , as well as a weak band of m r 55 kda corresponding to full length pdgf - c . the results show that conditioned medium from the ag1523 fibroblasts produced proteolytic activity that will process full length pdgf - c into active and receptor - competent pdgf - c . recombinant full - length human pdgf - c was expressed in sf9 insect cells using the baculovirus expression system ( see , e . g ., example 3 ; and li et al ., 2000 , nat . cell biol . 2 : 302 - 309 , incorporated herein by reference ). recombinant full - length pdgf - c is expressed as a major species of mr 55k in baculovirus - infected sf9 cells . serum - free medium was collected . tca - precipitated proteins from 0 . 2 ml of the medium were subjected to sds - page using a 12 % polyacrylamide gel ( biorad ) under reducing conditions and then immunoblotted . the his 6 - tagged pdgf - c was detected using an anti - his 6 epitope monoclonal antibody ( c - terminal , invitrogen ). no protein was detected in 1523 medium with this anti - his 6 epitope monoclonal antibody . bound antibodies were observed using enhanced chemiluminiscence plus ( ecl +; amersham ). as seen in fig3 , there is a light band at about 25 k , indicating a low but nonetheless significant endogenous processing of full length pdgf - c . further , it can be seen that his 6 epitopes in proteins in the medium are absent from ag1523 cells . to elucidate the mechanism of the proteolysis of pdgf - c a protease inhibitor analysis was conducted . various protease inhibitors ( see table 1 , source : sigma ) were pre - incubated with 0 . 9 ml of ag1523 serum - free medium at room temperature for 30 minutes , then incubated with 0 . 2 ml of recombinant full - length pdgf - c ( sf9 serum - free medium ) at 37 ° c . overnight . tca - precipitated proteins were subjected to sds - page under reducing conditions and then immunoblotted . recombinant pdgf - c was detected using an anti - his 6 epitope monoclonal antibody ( c - terminal ) ( invitrogen ). by increasing the amount of conditioned ag1523 medium and varying the co - incubated protease inhibitors , recombinant full - length pdgf - cc was cleaved in a dose - dependent manner . this indicates that the involved protease is present in the ag1523 medium and that the processing occurs extracellularly . the serine protease inhibitors were able to decrease the proteolysis as compared to control , indicating the serine proteases are those involved in the processing of pdgf - c . in particular , aprotinin showed a capacity to inhibit proteolytic processing , thus the serine protease is expected to be trypsin - like . trypsin - like serine proteases are proteases containing trypsin like domains . as seen in corresponding fig3 , conditioned medium from ag1523 fibroblasts contains a serine protease with trypsin - like properties that processes pdgf - c . chronic myocardial ischemia was replicated by ligation of the left anterior descending ( lad ) coronary artery using anesthetized 10 week old normal c57b16 mice . for pdgf - c treatment mice , 10 μg of recombinant human pdgf - cc core domain protein produced in baculovirus infected insect cells were administered after heart infarction using a subcutaneous osmotic minipump for seven days ( alzet ™ osmotic pump , durect corporation , cupertino , calif .). seven days after lad ligation , infarcted hearts were fixed and collected . the pdgf - cc core domain protein ( seq id no : 40 ) corresponds to corresponds to residues 230 - 345 of full - length pdgf - c protein i . e . amino acids 230 - 345 of seq id no : 3 . the hearts were sectioned longitudinally into 6 μm sections . hematoxylin - eosine and immunohistochemical stainings were performed using thrombomodulin as a marker for endothelial cells . smooth muscle alpha - actin was used as a marker for vascular smooth muscle cells . infarcted areas and vessel densities were calculated using a quantinet q600 image analysis system ( leica , brussels , belgium ). data were statistically analyzed using the student t test . in the pdgf - cc treated mice , total vessel density was about 136 % of that of the normal mice ( p = 0 . 07 , 56 ± 16 . 6 versus 41 . 2 ± 14 . 2 total vessels / mm 2 ). values are presented as the average ± sd , pdgf - cc treated mice n = 6 versus normal mice n = 11 . the vessels were further classified into three different groups , large (& gt ; 30 μm ), medium ( 10 - 30 μm ), and small (& lt ; 10 μm ). the large vessel density in pdgf - cc treated mice was 114 % of that of the normal ( untreated ) mice ( p = 0 . 48 , 8 . 3 ± 3 . 2 versus 7 . 3 ± 2 . 5 large vessels / mm 2 ). the medium vessel density in pdgf - cc treated mice was 111 % of that of the normal ( untreated ) mice ( p = 0 . 53 , 14 . 5 ± 3 . 7 versus 13 ± 4 . 7 medium vessels / mm 2 ). the small vessel density in pdgf - cc treated mice was 159 . 4 % of that of the normal ( untreated ) mice ( p = 0 . 038 , 33 . 2 ± 12 . 5 versus 20 . 8 ± 9 . 7 small vessels / mm 2 ). [ 0215 ] fig3 shows smooth muscle actin ( sma ) staining in normal ( a ) and pdgf - cc treated ( b ) hearts after infarction . the smooth muscle cell marker stains smooth muscle cells surrounding the vessels . in the infarcted area of the pdgf - cc treated mice ( b ), there are more positive stainings of small sized vessels compared with those in the infarcted area of untreated hearts ( a ). [ 0216 ] fig3 shows average data for vessel densities in the infarcted area . all vessel sizes showed increased presence in the pdgf - cc treated mice . the difference in small vesels was statistically significant ( p = 0 . 038 ). data are presented as average ± standard deviation ( sd ). open bars represent non - treated , and solid bars represent treated groups . the same experiment as discussed in example 16 was repeated using 30 μg recombinant pdgf - c per mouse . the results are shown in fig3 and 35 . [ 0218 ] fig3 shows capillary density in the infarcted area 7 days following the induction of myocardial infarction in mice , treated ( solid bars ) or un - treated ( open bars ) with 30 μg of recombinant pdgf - c delivered via a mini - osmotic pump . [ 0219 ] fig3 shows the density of smooth muscle α - actin coated vessels in the infarcted area 7 days following the induction of myocardial infarction in mice , treated ( solid bars ) or un - treated ( open bars ) with 30 μg of recombinant pdgf - c delivered via a mini - osmotic pump . total thrombomodulin positive vessels in pdgf - c treated mice had a density 151 % of that of normal ( untreated ) mice . the density of large , medium , small vessels in pdgf - cc treated mice are 167 %, 153 %, and 147 %, respectively , of those of normal ( untreated ) mice . total sma positive vessels in pdgf - cc treated mice had a density 141 % of that of normal ( untreated ) mice . the density of large , medium , small vessels are 114 %, 142 %, and 145 % respectively , of those of normal ( untreated ) mice . the results showed that treatment with 30 μg per mouse over the 7 days significantly stimulated revascularization of the infarcted area , and the stimulation was more significant than treatment with 10 μg per mouse . all vessel types seemed to respond to the treatment . combined with the data shown in example 16 , these example shows that pdgf - c stimulates revascularization of infarcted areas in a dose - dependent manner , and supports the conclusion that pdgf - c is useful in treating myocardinal ischemia . acute myocardial ischemia and hind limb ischemia mouse models were performed as previously described ( luttun et al ., nat med , 2002 1 : 1 ; heymans et al ., nat med , 1999 5 : 1135 - 42 ). subcutaneously implanted osmotic minipumps ( alzet , type 2001 ) were used for continuous protein delivery for 7 days . human pdgf - cc core domain protein was produced as described ( li et al ., nat cell biol , 2000 2 : 302 - 309 ). fluorescent or color dye microspheres ( yellow , 15 μm , molecular probes ) were administered after maximal vasodilatation ( sodium nitroprusside , 50 ng / ml , sigma ) for blood flow measurement , and flow was calculated as described ( carmeliet et al ., nat med , 1999 5 : 495 - 502 ). for histology , the hearts were harvested seven days after lad ligation , and sectioned longitudinally ( 6 μm ). infarcted areas were morphologically inspected after immunohistochemistry staining using thrombomodulin ( rabbit anti - tm , for all vessels ) and smooth muscle alpha - actin ( mouse anti - sma , for mature smc covered vessels , dako ), and vessel densities calculated . gastrocnemius muscles after femoral artery ligation were sectioned transversally and analysed after h & amp ; e or immunostainings with the ec marker cd31 ( pecam , rat anti - cd31 , pharmingen ). vessel densities and tissue necrosis / regeneration in the gastrocnemius muscle were analyzed morphometrically using the ks300 image analysis soft ware ( zeiss ). remodeling of collateral vessels in the upper hindlimb after femoral ligation was quantified as reported ( luttun et al ., nat med , 2002 1 : 1 ). for epc mobilization assay , mice were treated with pdgf - cc protein ( 4 . 5 μg / day ) immediately after femoral artery ligation using subcutaneously implanted osmotic minipumps ( alzet , type 2001 ). after two or five days , mice were sacrificed and spleens harvested for epc analysis using procedures described previously ( asahara et al ., circ res , 1999 85 : 221 - 8 ; dimmeler et al ., j . clin . invest ., 2001 108 : 391 - 397 ). spleens were mechanically minced using syringe plungers and laid over ficoll to isolate splenocytes . splenocytes were seeded into fibronectin - coated 24 - well plates in 0 . 5 ml ebm medium . after three weeks of culturing , adherent cells were stained for dii - ac - ldl / lectin and number of the positive cells counted . late outgrowth epcs ( after 3 weeks of culture ) were identified by metabolic uptake of dii - acetylated - ldl ( molecular probes ) and positive staining of alexa 488 - labeled isolectin b4 ( molecular probes ). quantification of the epc density was performed by confocal microscopy in five microscopic fields at 200 × magnification , and average epc density calculated . enriched human bm derived ac133 + cd34 + cells ( clonetics ) at 10 5 / ml were cultured for 3 days in hpgm ( clonetics ) in a 6 - well plate ( becton dickinson ). cells were then seeded in collagen coated 12 - well plates in ebm ( clonetics ) medium containing 4 % fcs and vegf 165 ( r & amp ; d systems ) or pdg - fcc ( 50 ng / ml each ). growth factors were added every two days and media were refreshed at 75 % every four days . for adherence assay , 2 . 5 × 10 4 of non - adherent cells / ml were cultured in the same condition on chamber slides coated with collagen , or in 96 - well plate coated with 0 . 3 % gelatin in pbs . cells were then washed three times with pbs , fixed and stained with may - grüinwald giemsa ( sigma ) after two weeks of culture on chamber slides ( becton dickinson ). the number of viable cells was estimated by atp quantification using celltiter - glo luminescent cell viability assay ( promega ) according to the manufacturer &# 39 ; s instructions . for cell surface marker staining , cells ( 2 × 10 4 / well ) cultured on collagen - coated culture slides for two or four weeks were fixed ( 45 min , 25 ° c .) and permeabilized ( 45 min , 25 ° c .) using a intrastain kit ( dako ), and then labeled with cd31 - fitc ( becton dickinson ), cd144 - fitc ( pharmingen ), cd34 - fitc ( becton dickinson ) or smc - actin cy3 ( sigma ). single or double - labeled cells were analyzed using laser confocal immunofluorescence microscopy . cell migration assays were performed on growth arrested confluent hmvec , baec or hsmc cells . cell monolayers were wounded with a rubber policeman and washed with serum - free medium . dishes were then incubated for 20 hours in serum - free medium containing vegf 165 , pdgf - aa , ( r & amp ; d systems , minneapolis usa ) or pdgf - cc . each assay included two dishes per condition and was repeated three times independently . cells were photographed at 40 × magnification , and migration percentage corresponding to the ratio between area of the cells and the total area of the wound ( biocom visiol @ b 2000 version 4 . 52 , san diego ). for hmvec proliferation assay , cells were seeded in 96 - well plates ( 5 wells per condition ), and incubated with vegf , pdgf - aa , or pdgf - cc ( 50 ng / ml ) after serum starvation . after 7 days , viable cells were counted using the cell titer - glo luminescent cell viability assay . for nih - 3t3 and hsmc proliferation assay , cells cultured in 96 - well plates were serum - starved overnight , followed by treatment with growth factors at different concentrations . two days later , cell numbers were counted and proliferation percentage calculated , using cells cultured in medium containing 10 % serum as control . aortic ring assay was performed as described 51 . briefly , one - millimeter long aortic rings were embedded in gels of rat tail interstitial collagen and cultured at 37 ° c ., supplemented with different growth factors ( 50 ng / ml ). aortic rings were analysed at day 9 of culturing . experiments included three explants per condition and were repeated at least twice . aortic rings were photographed at 25 × magnification . rnase protection analysis ( rpa ) was performed according to the manufacturer &# 39 ; s protocol ( ambion ) to investigate gene expression at mrna level . riboprobes were prepared using rna polymerase ( promega ) and 32 p - utp ( amersham ). mouse β - actin cdna ( 250 bp , ambion ) was used as an internal control . for western blot assay , subconfluent cells were rinsed with cold pbs supplemented with 5 g / ml of antiprotease cocktail , lysed in ripa buffer and analyzed on 10 % acrylamide sds page in reducing condition . two antibodies to pdgfr - α ( rabbit polyclonal antibody , dilution : 1 / 500 , santa cruz , sc431 ; and monoclonal peroxidase - labeled anti - rabbit antibody , dilution : 1 / 2500 , sigma , a2074 ) were used for pdgfr - α protein detection . membranes were developed using the supersignal system ( pierce ). for receptor activation , tissue / cell lysates were subjected to immunoprecipitation using the rabbit anti - pdgfr - α antibody . the precipitants were analysed on sds - page , and immunoblotted using a monoclonal anti - phosphotyrosine antibody ( santa cruz ). for pdgf - c over - expression , mouse full - length pdgf - c cdna was cloned into pcdna3 . 1 / zeo (+) mammalian expression vector ( invitrogen ) and the construct was verified by sequencing . plasmid dna was transfected into semiconfluent cells using lipofectamine plus reagent according to manufacturers protocol ( life technology ). stable transfectants were selected with 700 μg ml − 1 zeocin ( invitrogen ) for 3 weeks . resistant colonies were pooled and maintained in medium supplemented with 300 μg ml − 1 zeocin . for pdgf - cc western blot assay , cells were starved in serum - free medium overnight . conditioned media ( overnight ) were collected and protein concentration determined . thirty - five μg protein was trichloroacetic acid ( tca ) precipitated and subjected to western blot using affinity purified polyclonal rabbit antibodies against pdgf - cc 20 . all the samples were in triplicates and the experiment was repeated twice . secreted vegf protein was quantified using the quantikine immunoassay kit ( r & amp ; d system ) according to the manufacturers protocol . two - tailed student t - test was used for data analysis , with p & lt ; 0 . 05 considered statistically significant . for cell migration assay , anova dunett &# 39 ; s test was used for data analyzing , with p & lt ; 0 . 05 considered statistically significant . a previously established mouse model of myocardial ischemia was used to assess whether pdgf - cc is capable of stimulating the revascularization of ischemic myocardium . after coronary ligation , new vessels revascularize the ischemic core from its surrounding border region . for pdgf - cc to stimulate new vessel growth , its receptor , pdgfr - α , should be expressed in the heart . by rnase protection analysis , pdgfr - α transcripts were detectable in the normal myocardium ( fig3 a ). moreover , immunoprecipitation and subsequent western blotting using an equal amount of protein extract revealed that pdgfr - α protein levels were significantly upregulated in the ischemic border zones surrounding the infarcts , i . e . where vessel growth is most active , as compared to the rest of the normal myocardium ( fig3 b , pdgfr - α ). pdgfr - α was , as assessed by western blotting of the phosphorylated tyrosine residues after immunoprecipitation , highly activated in the border zone surrounding the infarcts ( fig3 b , ptyr ). to examine whether pdgf - cc could stimulate revascularization of the ischemic myocardium , we delivered , using a minipump , continuously over one week after coronary ligation , recombinant human pdgf - cc core domain protein , which is known to bind and activate pdgfr - α ( li et al ., nat cell biol , 2000 2 : 302 - 309 ). compared to control , pdgf - cc indeed increased the amount of active pdgfr - α in the border region ( fig3 b ). after seven days , angiogenesis was quantified by counting the number of endothelial cell ( ec )- lined vessels in the ischemic area after immunolabeling with thrombomodulin ( tm ). vessel maturation ( arteriogenesis ) was evaluated by counting the arterioles , immunoreactive for smooth muscle cell α - actin ( sma ). at 1 . 5 μg / day , pdgf - cc minimally affected the tm - positive vessel density ( fig3 c , e , f ) but increased , by 1 . 36 - fold , the number of sma - positive arterioles ( fig3 d , h , i ) ( sma positive vessels / mm 2 : 53 . 1 ± 3 . 7 after pdgfc vs 38 . 6 ± 4 . 8 after saline , n = 15 , 16 , p = 0 . 02 ). when using a 3 - fold higher dose ( 4 . 5 μg / day ), pdgf - cc significantly stimulated angiogenesis ( fig3 c , e , g ) and arteriogenesis ( fig3 d , h , j ). no signs of hemorrhage , edema or fibrosis were observed in the pdgf - cc treated hearts . these new vessels were functional as perfusion of the ischemic myocardial region was significantly increased ( blood flow : 1 . 6 ± 0 . 2 ml / min / g in control versus 2 . 2 ± 0 . 2 ml / min / g after 4 . 5 μg / day pdgf - cc ; n = 7 - 9 ; p & lt ; 0 . 05 ). the effect of pdgf - cc to stimulate revascularization appeared to be restricted to the ischemic heart , as no differences were observed in vessel density in other organs ( not shown ). the magnitude of the potential of pdgf - cc to stimulate revascularization of the ischemic myocardium parallels that of vegf and plgf ( luttun et al ., nat med , 2002 1 : 1 ). the mice tolerated the pdgf - cc treatment without problems , appeared healthy and had no signs of toxicity ( weight loss , inactivity ). thus , pdgf - cc protein treatment promoted functional revascularization in cardiac ischemia via enhanced angiogenesis ( more vessels ) and arteriogenesis ( more smc coverage ). the angio / arteriogenic activity of pdgf - cc in cardiac ischemia is remarkable , since the other pdgfr - α ligand , pdgf - aa is poorly angiogenic or even suppresses angiogenesis ( de marchis et al ., blood , 2002 99 : 2045 - 2053 ; palumbo et al ., arterioscler thromb vasc biol , 2002 22 : 405 - 11 ; koyama et al ., j cell physiol , 1994 158 : 1 - 6 ). to further verify the angio / arteriogenic activity of pdgf - cc in vivo , the effect of pdgf - cc in an established mouse model of hind limb ischemia is investigated ( luttun et al ., nat med , 2002 1 : 1 ). pdgfr - α expression was quantified by rnase protection analysis in the gastrocnemius muscle , which becomes highly ischemic after ligation of the femoral artery ( deindl et al ., circ res , 2001 89 : 779 - 86 ; couffinhal et al ., american journal of pathology , 1998 152 : 1667 - 1679 ). two days after femoral artery ligation , when a fraction of myocytes died due to ischemic necrosis , pdgfr - α transcript levels decreased to 76 % of those found in normal muscles ( fig3 a ). however , compared to vehicle , a daily treatment with 4 . 5 μg pdgf - cc upregulated pdgfr - α expression at day 2 after ligation and almost completely restored its expression levels to those found in the unligated control muscle ( fig3 a ). revascularization of the ischemic gastrocnemius muscle , which only occurred in those regions where regenerating muscle replaced the necrotic avascular muscle , was scored after continuous delivery , by osmotic minipump , of 4 . 5 μg pdgf - cc per day for one week after femoral artery ligation . treatment with pdgf - cc after femoral artery ligation not only increased angiogenesis ( e . g . the capillary density ; fig3 b , f , g ), it also enhanced arteriogenesis ( e . g . the density of sma + vessels ; fig3 c ). moreover , pdgf - cc enhanced skeletal muscle regeneration ( fig2 e , h - l ) and , as a result , also reduced the extent of ischemic muscle necrosis ( fig3 d , h - l ), suggesting that muscle regeneration and angiogenesis might be linked . pdgf - cc also enlarged the second - generation collateral side branches in the adductor muscle ( 680 ± 40 μm 3 after saline versus 920 ± 100 μm 3 after pdgf - cc ; n = 10 ; p = 0 . 05 ). no signs of hemorrhage , edema or fibrosis were observed in the pdgf - cc treated limbs . thus , pdgf - cc stimulates revascularization in mouse models of both heart and limb ischemia . to examine the possible mechanism of how pdgf - cc stimulates vessel growth and maturation , we next assessed its effects on vascular endothelial progenitor cells ( epcs ). several vascular growth factors , such as vegf and plgf , have been shown to mobilize vascular stem / progenitor cells to sites of vessel growth and tissue repair (( kalka et al ., ann thorac surg , 2000 70 : 829 - 34 ; hattori et al ., nat med , 2002 1 : 1 ; rafii et al ., semin cell dev biol , 2002 13 : 61 - 7 ; asahara et al ., embo j , 1999 18 : 3964 - 72 ; carmeliet et al ., thromb haemost , 2001 86 : 289 - 97 ). a possible role of pdgf - cc in epc mobilization has , however , not been investigated thus far . we quantified epc mobilization by counting the number of acldl - dii / isolectin - ib4 positive endothelial cells after 3 weeks of plating spleen mononuclear cells . by scoring after 3 weeks , only late - outgrowth epcs , but not surviving sloughed - off endothelial cells , are selectively assayed ( lin et al ., j clin invest , 2000 105 : 71 - 7 ; rafii , s ., j clin invest , 2000 105 : 17 - 9 ). in baseline conditions , pdgf - cc did not affect mobilization of epcs ( fig3 a ), consistent with our observation that pdgf - cc did not affect vessel growth in non - ischemic organs but only in ischemic tissues ( see above ). we therefore ligated the femoral arteries and found that treatment with pdgf - cc for two days ( 4 . 5 μg / day via minipump ) augmented epc mobilization approximately 3 - fold above the levels found in the control group ( fig3 a - g ). this augmentation persisted to day five , albeit at a lower level ( fig3 a ). thus , pdgf - cc treatment enhanced epc mobilization in tissue ischemia , thereby providing a source of ecs needed for revascularization of ischemic tissues . pdgf - cc enhances differentiation of bone marrow progenitors into both ecs and smcs upon stimulation by growth factors or cytokines , bone marrow stem / progenitor cells can differentiate into ecs and smcs and thereby contribute to angio / arteriogenesis ( orlic et al ., nature , 2001 410 : 701 - 5 ; kawamoto et al ., circulation , 2001 103 : 634 - 7 ; asahara et al ., circ res , 1999 85 : 221 - 8 ). the potential role of pdgf - cc in the differentiation of bone marrow progenitors into vascular cells has , however , not been investigated thus far . we therefore cultured human bone marrow - derived ac133 + cd34 + cells — a population enriched for stem / progenitor cells ( miraglia et al ., blood , 1997 90 : 5013 - 21 ; yu et al ., j biol chem , 2002 277 : 20711 - 6 ; donnelly et al ., leuk lymphoma , 2001 40 : 221 - 34 )— and stimulated them with pdgf - cc , using vegf as a control ( 50 ng / ml each ). these cells expressed pdgfr - α , when analyzed by rt - pcr ( not shown ). after two weeks of stimulation , both pdgf - cc and vegf enhanced the adherence of these cells — a prerequisite for anchorage - dependent cell proliferation , differentiation , migration and prevention of apoptosis ( assoian , j cell biol , 1997 136 : 1 - 4 ; asahara et al ., science , 1997 275 : 964 - 7 ) ( fig3 a - d ). however , the two growth factors markedly differed in their ability to induce the commitment of these stem / progenitor cells into either the endothelial or smooth muscle cell lineage . after two weeks of stimulation , both pdgf - cc and vegf induced the expression of the ec surface markers cd144 ( ve - cadherin ; fig3 e - g ) and cd31 ( pecam ; fig3 h - j ), indicating that these growth factors induced a characteristic endothelial phenotype . interestingly , only pdgf - cc additionally induced the expression of the smooth muscle cell marker sma in a fraction of these cells , indicating that these cells had acquired a characteristic smc phenotype ( fig3 k - m ). notably , this pleiotropic effect of pdgf - cc in inducing both endothelial and smooth muscle lineages was specific , as the vegf - treated cells did not become sma positive ( fig3 k - m ). double labeling experiments revealed that pdgf - cc often induced the expression of cd31 and sma in the same cells . by four weeks , most (& gt ; 95 %) of the pdgf - cc - treated cells were sma positive and had lost their expression of cd144 and cd31 , while vegf - treated cells , instead , were still cd144 and cd31 positive but remained sma negative ( not shown ). thus , pdgf - cc initially induced bone marrow progenitor cells to differentiate into cell types with both endothelial or smooth muscle cell characteristics — eventually , after long - term treatment , yielding cells with a smc - like phenotype . pdgf - cc thus differed from vegf , as the latter only caused bone marrow progenitors to acquire ec - specific markers , even after prolonged treatment . although pdgfr - α is expressed on endothelial cells ( edelberg et al ., journal of clinical investigation , 1998 102 : 837 - 43 ; smits et al ., growth factors , 1989 2 : 1 - 8 ; bar et al ., endocrinology , 1989 124 : 1841 - 8 ; beitz et al ., proc natl acad sci usa , 1991 88 : 2021 - 5 ; marx et al ., j clin invest , 1994 93 : 131 - 9 ; shinbrot et al ., dev . dyn ., 1994 199 : 169 - 175 ), little is known about the functional consequence of pdgfr - α signaling in these cells . we therefore compared the effect of pdgf - cc on ec migration and proliferation to that of vegf ( which primarily affects endothelial cells ( senger et al ., am j pathol , 1996 149 : 293 - 305 ) and pdgf - aa ( which primarily affects fibroblasts and smooth muscle cells ( heldin et al ., physiol rev , 1999 79 : 1283 - 1316 )). pdgfr - a expression on the human microvascular endothelial cells ( hmvec ) was confirmed by western blot , albeit at a lower level as compared with that of the smcs ( not shown ). vegf and pdgf - cc , but not pdgf - aa , stimulated migration of hmvecs ( fig4 a ). this effect of pdgf - cc was not restricted to hmvecs only , as pdgf - cc also enhanced the migration of bovine aorta endothelial cells ( baec ; fig4 b ). however , none of the pdgfs affected ec proliferation ( fig4 c ), in agreement with the previous observation that pdgfr - α does not transmit mitogenic signals in response to pdgf - aa in ecs ( marx et al ., j clin invest , 1994 93 : 131 - 9 ). vegf , instead , highly stimulated ec proliferation ( fig5 c ). we also tested the effect of pdgf - cc on cultured aortic rings , as this assay allows assessment of the outgrowth of microvessels from an intact vessel in vitro ( blacher et al ., angiogenesis , 2001 4 : 133 - 42 ). results are graphically represented as the number of microvessels and the distance over which they grew out from the aortic ring . each experiment included three explants per condition and was repeated at least twice . at day 9 after culturing , microvessels and the distance of their outgrowth were quantified and evaluated using student &# 39 ; s t test . in baseline conditions , only a small number of microvessels sprouted from the aortic rings — most of them over very short distances ( 0 . 25 mm from the aortic ring )— and only a small fraction (& lt ; 5 %) growing out over longer distances (& gt ; 0 . 5 mm from the aortic ring , fig4 a ). vegf increased not only the number of sprouting microvessels ( p & lt ; 0 . 001 at all concentrations versus control ), but also the distance over which they grew out ( p & lt ; 0 . 05 at all concentrations versus control ; fig4 b , f ). at 30 ng / ml , pdgf - cc increased the number of microvessels ( p & lt ; 0 . 001 versus control , fig4 e , g ) and increased the distance of vessel outgrowth at 5 ng / ml ( p & lt ; 0 . 01 versus control , fig4 g ). apparently , pdgf - cc had its maximum effect at 30 ng / ml on microvessel sprouting , and was less potent at a concentration of 50 ng / ml , indicating that the dose - response relationship of pdgf - cc in the aortic ring assay was bell - shaped . a similar bell - shaped dose - response relationship has been documented for other members of the vegf / pdgf - superfamily ( jin et al ., j mol neurosci , 2000 14 : 197 - 203 ). pdgf - aa , however , had no effect on the number of microvessels , although it increased the distance of vessel outgrowth at 5 ng / ml ( p & lt ; 0 . 01 versus control , fig4 h ). thus , pdgf - cc mobilized ec migration in cultured cells and promoted microvessel sprouting in the aortic ring assay . this chemotactic effect of pdgf - cc on ecs is surprising , since although the other pdgfs are among the most potent stimuli of mesenchymal cell migration , they do not or minimally stimulate — and , in certain conditions , even inhibit — ec migration ( de marchis et al ., blood , 2002 99 : 2045 - 2053 ). pdgf - cc is both chemotactic and mitogenic for smcs and perivascular fibroblast cells the mitogenic and chemotactic effect of pdgf - cc was then tested on smcs and perivascular fibroblast cells , and the effect of pdgf - cc was compared to that of vegf and pdgf - aa in both cultured cells and the aortic ring assay ( blacher et al ., angiogenesis , 2001 4 : 133 - 42 ). pdgf - cc - treated cultured hsmc and nih - 3t3 fibroblast cells expressed significant amounts of pdgfr - α ( fig4 a , pdgfr - α ). subsequent immunoblotting for ptyr indicated that pdgfr - α was highly activated ( fig4 a , ptyr ). both pdgf - cc and - aa stimulated hsmc migration with a comparable potency , while vegf had no effect ( fig4 b ). pdgf - cc and - aa also stimulated the proliferation of cultured nih - 3t3 fibroblast and hsmc cells , the effect of pdgf - cc on the latter cells being slightly more pronounced ( fig4 c , d ). in the aortic ring assay , we quantified the growth and emigration of perivascular fibroblasts from the intact vessel using computer - assisted image analysis after treatment with vegf and different pdgfs at different concentrations . in baseline conditions , individual perivascular fibroblast - like cells ( identified as isolated cells , not associated with sprouting microvessels ) were sparse and emigrated over only short distances from the aortic ring ( fig4 a ). while vegf was ineffective on the perivascular fibroblast - like cells ( fig4 b , i ), pdgf - cc significantly increased the number of such cells , which also emigrated over much greater distances from the aortic ring ( p & lt ; 0 . 001 at all concentrations versus control , fig4 c - e , j ). at high concentrations ( 30 - 50 ng / ml ), pdgf - cc still stimulated fibroblast - like cell growth and emigration but less significantly than at lower concentrations , possibly because its effects were dose - dependent ( see above ) and / or the perivascular cells surrounded the sprouting microvessels . pdgf - aa had an intermediate effect on the perivascular fibroblast - like cells ( p & lt ; 0 . 05 at different concentrations versus control , fig4 k ). thus , pdgf - cc , more potently than pdgf - aa , stimulated the migration and proliferation of perivascular cells in the aortic ring assay — an assay that is believed to reflect more closely the in vivo situation and allows synergistic interactions between the different vascular cell types ( hartlapp et al , 2001 , faseb j , 15 : 2215 - 24 ). assays are conducted to evaluate whether pdgf - c has similar activities to pdgf - a , pdgf - b , vegf , vegf - b , vegf - c and / or vegf - d in relation to growth and / or motility of connective tissue cells , fibroblasts , perivascular , myofibroblasts and glial cells ; to endothelial cell function ; to angiogenesis ; and to wound healing . further assays may also be performed , depending on the results of receptor binding distribution studies . to test the mitogenic capacity of pdgf - c for endothelial cells , the pdgf - c polypeptide is introduced into cell culture medium containing 5 % serum and applied to bovine aortic endothelial cells ( baes ) propagated in medium containing 10 % serum . the baes are previously seeded in 24 - well dishes at a density of 10 , 000 cells per well the day before addition of the pdgf - c . three days after addition of this polypeptide the cells were dissociated with trypsin and counted . purified vegf is included in the experiment as positive control . endothelial cell growth assays are performed by methods well known in the art , e . g . those of ferrara & amp ; henzel , nature , 1989 380 : 439 - 443 , gospodarowicz et al ., proc . natl . acad . sci . usa , 1989 86 : 7311 - 7315 , and / or claffey et al ., biochem . biophys . acta , 1995 1246 : 1 - 9 . the effect of pdgf - c on adhesion of polymorphonuclear granulocytes to endothelial cells is tested . the standard boyden chamber chemotaxis assay is used to test the effect of pdgf - c on chemotaxis . endothelial cells are tested for the effect of pdgf - c on plasminogen activator and plasminogen activator inhibitor production , using the method of pepper et al ., biochem . biophys . res . commun ., 1991 181 : 902 - 906 . the ability of pdgf - c to stimulate endothelial cells to migrate and form tubes is assayed as described in montesano et al ., proc . natl . acad . sci . usa , 1986 83 : 7297 - 7301 . alternatively , the three - dimensional collagen gel assay described in joukov et al ., embo j ., 1996 15 : 290 - 298 or a gelatinized membrane in a modified boyden chamber ( glaser et al ., nature , 1980 288 : 483 - 484 ) may be used . the ability of pdgf - c to induce an angiogenic response in chick chorioallantoic membrane is tested as described in leung et al ., science , 1989 246 1306 - 1309 . alternatively the rat cornea assay of rastinejad et al ., cell , 1989 56 345 - 355 may be used ; this is an accepted method for assay of in vivo angiogenesis , and the results are readily transferrable to other in vivo systems . the ability of pdgf - c to stimulate wound healing is tested in the most clinically relevant model available , as described in schilling et al ., surgery , 1959 46 : 702 - 710 and utilized by hunt et al ., surgery , 1967 114 : 302 - 307 . a variety of in vitro and in vivo assays using specific cell populations of the haemopoietic system are known in the art , and are outlined below . in particular a variety of in vitro murine stem cell assays using fluorescence - activated cell sorter to purified cells are particularly convenient : these are cells capable of repopulating the bone marrow of lethally irradiated mice , and have the lin − , rh h1 , ly - 6a / e + , c - kit + phenotype . pdgf - c is tested on these cells either alone , or by co - incubation with other factors , followed by measurement of cellular proliferation by 3 h - thymidine incorporation . these are cells that have comparatively little bone marrow repopulating ability , but can generate d13 cfu - s . these cells have the lin − , rh h1 , ly - 6a / e + , c - kit + phenotype . pdgf - c is incubated with these cells for a period of time , injected into lethally irradiated recipients , and the number of d13 spleen colonies enumerated . these are cells that respond in vitro to single growth factors and have the lin − , rh h1 , ly - 6a / e + , c - kit + phenotype . this assay will show if pdgf - c can act directly on haemopoietic progenitor cells . pdgf - c is incubated with these cells in agar cultures , and the number of colonies present after 7 - 14 days is counted . smooth muscle cells play a crucial role in the development or initiation of atherosclerosis , requiring a change of their phenotype from a contractile to a synthetic state . macrophages , endothelial cells , t lymphocytes and platelets all play a role in the development of atherosclerotic plaques by influencing the growth and phenotypic modulations of smooth muscle cell . an in vitro assay using a modified rose chamber in which different cell types are seeded on to opposite cover slips measures the proliferative rate and phenotypic modulations of smooth muscle cells in a multicellular environment , and is used to assess the effect of pdgf - c on smooth muscle cells . the ability of pdgf - c to inhibit metastasis is assayed using the lewis lung carcinoma model , for example using the method of cao et al ., j . exp . med ., 1995 182 : 2069 - 2077 . the effects of the pdgf - c on the migration of smooth muscle cells and other cells types can be assayed using the method of koyama et al ., j . biol . chem ., 1992 267 : 22806 - 22812 . the effects of the pdgf - c on chemotaxis of fibroblast , monocytes , granulocytes and other cells can be assayed using the method of siegbahn et al ., j . clin . invest ., 1990 85 : 916 - 920 . the effects of pdgf - c on proliferation , differentiation and function of other cell types , such as liver cells , cardiac muscle and other cells , endocrine cells and osteoblasts can readily be assayed by methods known in the art , such as 3 h - thymidine uptake by in vitro cultures . expression of pdgf - c in these and other tissues can be measured by techniques such as northern blotting and hybridization or by in situ hybridization . pdgf - c is a member of the pdgf family of growth factors which exhibits a high degree of homology to the other members of the pdgf family . pdgf - c contains eight conserved cysteine residues which are characteristic of this family of growth factors . these conserved cysteine residues form intra - chain disulfide bonds which produce the cysteine knot structure , and inter - chain disulfide bonds that form the protein dimers which are characteristic of members of the pdgf family of growth factors . pdgf - c interacts with a protein tyrosine kinase growth factor receptor . in contrast to proteins where little or nothing is known about the protein structure and active sites needed for receptor binding and consequent activity , the design of active mutants of pdgf - c is greatly facilitated by the fact that a great deal is known about the active sites and important amino acids of the members of the pdgf family of growth factors . published articles elucidating the structure / activity relationships of members of the pdgf family of growth factors include for pdgf : oestman et al ., j . biol . chem ., 1991 266 : 10073 - 10077 ; andersson et al ., j . biol . chem ., 1992 267 : 11260 - 1266 ; oefner et al ., embo j ., 1992 11 : 3921 - 3926 ; flemming et al ., molecular and cell biol ., 1993 13 : 4066 - 4076 and andersson et al ., growth factors , 1995 12 : 159 - 164 ; and for vegf : kim et al ., growth factors , 1992 7 : 5364 ; pötgens et al ., j . biol . chem ., 1994 269 : 32879 - 32885 and claffey et al ., biochem . biophys . acta , 1995 1246 : 1 - 9 . from these publications it is apparent that because of the eight conserved cysteine residues , the members of the pdgf family of growth factors exhibit a characteristic knotted folding structure and dimerization , which result in formation of three exposed loop regions at each end of the dimerized molecule , at which the active receptor binding sites can be expected to be located . based on this information , a person skilled in the biotechnology arts can design pdgf - c mutants with a very high probability of retaining pdgf - c activity by conserving the eight cysteine residues responsible for the knotted folding arrangement and for dimerization , and also by conserving , or making only conservative amino acid substitutions in the likely receptor sequences in the loop 1 , loop 2 and loop 3 region of the protein structure . the formation of desired mutations at specifically targeted sites in a protein structure is considered to be a standard technique in the arsenal of the protein chemist ( kunkel et al ., methods in enzymol ., 1987 154 : 367 - 382 ). examples of such site - directed mutagenesis with vegf can be found in pötgens et al ., j . biol . chem ., 1994 269 : 32879 - 32885 and claffey et al ., biochem . biophys . acta , 1995 1246 : 1 - 9 . indeed , site - directed mutagenesis is so common that kits are commercially available to facilitate such procedures ( e . g . promega 1994 - 1995 catalog ., pages 142 - 145 ). the connective tissue cell , fibroblast , myofibroblast and glial cell growth and / or motility activity , the endothelial cell proliferation activity , the angiogenesis activity and / or the wound healing activity of pdgf - c mutants can be readily confirmed by well established screening procedures . for example , a procedure analogous to the endothelial cell mitotic assay described by claffey et al ., biochem . biophys . acta ., 1995 1246 : 1 - 9 can be used . similarly the effects of pdgf - c on proliferation of other cell types , on cellular differentiation and on human metastasis can be tested using methods which are well known in the art . the foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting . since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art , the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof . arg arg gly thr gln ala glu ser asn leu ser ser lys phe gln phe ser ser asn lys glu gln asn gly val gln asp pro gln his glu arg glu glu asn val trp ile gln leu thr phe asp glu arg phe gly leu glu pro ser asp gly thr ile leu gly arg trp cys gly ser gly thr val ser asp glu tyr phe pro ser glu pro gly phe cys ile his tyr asn ile val met pro gln phe thr glu ala val ser pro ser val leu phe ser thr leu glu asp leu ile arg tyr leu glu pro glu arg trp leu thr glu glu val arg leu 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gly glu lys ile ile leu asn phe thr ser leu asp leu pro glu pro ile val ser thr asp ser arg leu trp val glu phe arg cys gly gly asp val lys lys asp tyr gly his ile gln ser pro asn tyr pro asp asp tyr arg pro ser lys val cys ile trp arg ile gln glu arg met asp ser cys ala tyr asp tyr leu glu val arg asp gly his ser glu ser ser thr leu ile gly arg tyr cys gly tyr glu lys pro asp asp ile lys ser thr ser ser arg leu trp leu lys phe val trp pro lys glu tyr pro pro asn lys asn cys ile trp gln leu val ala pro thr gln tyr arg ile ser leu gln phe asp phe phe glu thr glu gly asn asp val cys lys tyr asp phe val glu val arg ser gly leu thr ala asp ser lys leu his gly lys phe cys gly ser glu lys pro glu val ile thr ser gln tyr asn asn met arg val glu pro lys tyr pro his ser tyr his pro ser glu lys cys glu trp leu ile gln ala pro asp pro tyr gln arg ile met ile asn phe asn pro his phe val ser asp tyr glu thr his gly ala gly phe ser ile arg tyr glu cys ser gln asn tyr thr thr pro ser gly val ile lys ser pro gly phe pro glu lys tyr pro asn ser leu glu cys thr tyr ile val phe glu pro asp ser asn pro pro gly gly met phe cys arg tyr asp arg leu glu ile trp asp gly phe pro asp val gly pro his ile gly arg leu ser met val phe tyr thr asp ser ala ile ala lys glu gly phe cdna fragment encoding the cub domain which includes a bamhi site cdna fragment encoding the cub domain which includes a ecori site and sequences coding for a c - terminal 6x his tag preceded by an arg leu tyr ser cys thr pro arg asn phe ser val ser ile arg glu glu leu lys arg thr asp thr ile phe trp pro gly cys leu leu val cys gln cys val pro ser lys val thr lys lys tyr his glu val leu