Patent Application: US-85220901-A

Abstract:
pdgf - c , a new member of the pdgf / vegf family of growth factors , is described , as well as the nucleotide sequence encoding it , methods for producing it , antibodies and other antagonists to it , transfected and transformed host cells expressing it , pharmaceutical compositions containing it , and uses thereof in medical and diagnostic applications .

Description:
[ 0119 ] 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 , dc , 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 ′ forward ( seq id no : 25 ) 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 ). [ 0124 ] 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 . [ 0125 ] 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 . [ 0127 ] 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 . [ 0131 ] 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 . [ 0132 ] 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 . [ 0133 ] 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 . 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 ′ cg ggatcc cgaatccaacctgagtag3 ′ ( seq id no : 33 ). this primer includes a bamhi site ( underlined ) for in frame cloning . the reverse primer used was 5 ′ g gaattc ctaatggtgatggtgatgatgtttgtcatcgtcatctcctcctgtgctc cctct3 ′ ( seq id no : 34 ). 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 ) 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 ′ c ggatcc cggaagaaaatcca gagtggtg3 ′ ( seq id no : 35 ). this primer includes a bamhi site ( underlined ) for in frame cloning . the reverse primer used was 5 ′ g gaattc ctaatggtgatggtgatgatgtttgtcatcgtcatctcctcctgtg ctccctct - 3 ′ ( seq id no : 36 ). 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 . [ 0142 ] 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 . [ 0144 ] 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 mgcl 2 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 5 ′ cg ggatcc cgaatccaacctgagtag3 ′ ( seq id no : 38 ). this primer includes a bamhi site ( underlined ) for in clone frame cloning . the reverse primer used was 5 ′ ccg gaattc ctaatggtgatggtgatgatgtttgtcatcgtcgtcgacaatgttgta gtg3 ′ ( seq id no : 39 ). 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 . [ 0151 ] 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 . [ 0153 ] 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 ( 1 ). in fig2 i , 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 26 k ). 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 26 m ) and in the forming cleft between the lower lip buccal and the gingival epithelium ( arrows in 26 n ). 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 26 o and 26 p ). 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 mm , 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 1875 - 1887 ; soriano et al ., development , 1997 124 2691 - 70 ) were bred as c57bl6 / 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 { fraction ( 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 . recombinant human pdgf - cc core domain protein was expressed as described above ( cf . li et al ., nat cell biol 2000 2 302 - 9 ) and purified to homogeneity . two micrograms of the purified pdgf - cc were analyzed on a 4 - 12 % gradient bistris nupage ( norex ) polyacrylamide gel followed by staining with coomassie blue . the results are shown in fig2 . dimeric ( lane 2 ) and monomeric ( lane 3 ) forms of pdgf - cc were detected under non - reducing and reducing / alkylating conditions , respectively . molecular mass markers are indicated on the left ( lane 1 ). under non - reducing conditions the core domain of pdgf - cc appeared as dimers with the expected molecular mass of 31 kda ( lane 2 ). the dimeric forms of pdgf - cc were converted to monomers under reducing conditions in the presence of dtt ( lane 3 ). the chick embryo chorioallantoic membrane ( cam ) assay was performed according to previously published methods ( cao et al ., proc natl acad sci usa 1998 95 14389 - 94 ; cao et al ., proc natl acad sci usa 1999 96 5728 - 33 ). three - day - old fertilized white leghorn eggs ( ova production , sorgarden , sweden ) were cracked , and chick embryos with intact yolks were carefully placed in 20 × 100 mm plastic petri - dishes . after 6 days of incubation in 3 % co 2 at 37 ° c ., a disk of methylcellulose containing 2 . 5 μg of truncated pcgf - c homodimer ( pdgf - cc ) or bsa alone dried on a nylon mesh ( 3 × 3 mm ) was implanted on the cam of individual embryos . the nylon mesh disks were made by desiccation of 10 gl of 0 . 45 % methylcellulose in h 2 o . after 4 - 5 days of incubation , embryos and cams were examined for the formation of new blood vessels in the field of the implanted disks using a stereoscope . disks of methylcellulose containing 2 . 5 μg of bsa were used as negative controls . the experiments were carried out three times , and 9 embryos / sample were used for each experiment . the cam assay , which detects angiogenic activity of compounds during embryonic development , is one of the most widely used in vivo angiogenesis assays ( jain et al ., nat med 1997 3 1203 - 8 ). the early embryos in this angiogenesis assay avoid immune reactions and inflammatory influences on growing vessels . to demonstrate that pdgf - cc could induce angiogenesis in vivo , the core domain of pdgf - cc protein was implanted onto the chick chorioallantoic membrane in the developing embryo . nylon meshes ( 9 mm 2 ) coated with 0 . 45 % methylcellulose containing 2 . 5 μg of pdgf - cc or bsa were implanted on cams of 6 - day - old chick embryos . after 5 days of implantation , the formation of new blood vessels was examined under a stereoscope . fig3 a , 30b show a cam with a methylcellulose mesh containing bsa alone , which served as a negative control . fig3 c , 30d show an example of 2 . 5 μg of pdgf - cc - implanted cam . new blood vessels and sprouts are marked with arrows in fig3 c and 30d . it can be seen that pdgf - cc at the dose of 2 . 5 μg / disk was able to stimulate microvessel growth in each implanted chick embryo . a significant increase of neovascularization with a high vessel density was observed in the surrounding areas of pdgf - cc implant . notably , pdgf - cc induced the formation of new branches and induced vessel sprouts ( small arrows in fig3 c and 30d ) from the existing vessels that grew toward the implanted disks . these vessel sprouts appeared as “ red dots ” budding from blood vessels adjacent to the implanted factors . in contrast , disks without growth factors did not seem to stimulate neovascularization in chick embryos ( fig3 a , 30b ). the results clearly demonstrate that the truncated pdgf - c homodimer exhibits marked angiogenic activity in vivo . the mouse corneal micropocket assay was performed according to procedures previously described ( cao et al ., proc natl acad sci usa 1998 95 14389 - 94 ; cao et al ., nature 1999 398 381 ). male 5 - 6 week - old c57bi6 / j mice were acclimated and caged in groups of six or less . animals were anaesthetized by injection of a mixture of dormicum and hypnorm ( 1 : 1 ) before all procedures . corneal micropockets were created with a modified von graefe cataract knife in both eyes of each male 5 - 6 - week - old c57bi6 / j mouse . a micropellet ( 0 . 35 × 0 . 35 mm ) of sucrose aluminum sulfate ( bukh meditec , copenhagen , denmark ) coated with slow - release hydron polymer type ncc ( ifn sciences , new brunswick , n . j .) containing various amounts of truncated pdgf - c homodimer ( pdgf - cc ) was surgically implanted into each cornal pocket . for comparison purposes corresponding amounts of pdgf - aa , pdgf - ab , pdgf - bb , vegf 165 ( all obtained commercially from r & amp ; d systems of minnepolis , minn .) or fgf - 2 ( pharmacia & amp ; upjohn , milan , italy ) were similarly implanted into corneal pockets of test mice . in each case , the pellet was positioned 0 . 6 - 0 . 8 mm from the corneal limbus . after implantation , erythromycin / ophthalmic ointment was applied to each eye . on day 5 after growth factor implantation , animals were sacrificed with a lethal dose of co 2 , and corneal neovascularization was measured and photographed with a slit - lamp stereomicroscope . in fig3 and 32 , arrows point to the implanted pellets . the photographs represent 20 × amplification of the mouse eye . vessel length and clock hours of circumferential neovascularization were measured . quantitation of corneal neovascularization is presented as maximal vessel length ( fig3 e ), clock hours of circumferential neovascnlarization ( fig3 f ), and area of neovascularization ( fig3 g ). graphs represent mean values (± sem ) of 11 - 16 eyes ( 6 - 8 mice ) in each group . the corneal angiogenesis model is one of the most rigorous mammalian angiogenesis models that requires a putative compound to be sufficiently potent in order to induce neovascularization in the corneal avascular tissue . potent angiogenic factors including fgf - 2 and vegf have profound effects in this system . the angiogenic response of corneas stimulated by 160 ng of pdgf - cc was robust with a high number of capillaries ( fig3 b ). the newly formed as well as the limbal vessels were markedly dilated in the pdgf - cc - implanted corneas . the capillary vessel length of about 0 . 8 mm in corneas implanted with pdgf - cc was similar to that found in vegf - induced vessels ( fig3 b , 31d and 31 e ). the overall angiogenic response induced by pdgf - cc ( fig3 b ) was similar to that induced by fgf - 2 ( fig3 c ), albeit less potent than fgf - 2 . both pdgf - cc - and fgf - 2 - induced microvessels were well organized and separated ( fig3 b and 31c ). in contrast , the vegf - induced blood vessels ( fig3 d ) seemed to be leaky , hemorrhagic and likely to rupture . at the front edge , the vegf - induced capillaries were fused to into disorganized and sinusoidal structures . thus , angiogenic responses induced by pdgf - cc and vegf are markedly different from those induced by vegf but similar to those induced by fgf - 2 . the growth factor - implanted mouse eyes were enucleated at day 6 after implantation and immediately frozen on dry ice and stored at − 80 ° c . before use . tissue sections of 12 gm were dissected by a cryostat and were immersed in acetone for 10 min . tissue slices were washed with pbs , blocked with 30 % rabbit serum in pbs for 20 min . and incubated for 1 hour with a monoclonal rat anti - mouse antibody against cd31 antigen ( pharmingen ). after washing with pbs , a secondary fitc - conjugated rabbit anti - rat igg was incubated with the tissue sections for 1 hour . the immuno - stained signals were examined under a fluorescence microscope . corneal microvessels were counted in at least 6 sections at 20 × magnification . fig3 a - d show histological sections of pdgf - aa ( fig3 a ), pdgf - ab ( fig3 b ), pdgf - bb ( fig3 c ) and pdgf - cc ( fig3 d ) implanted corneas which were incubated with an anti - cd31 antibody and stained with a fitc - conjugated secondary antibody . microvessels are present in all sections . vessel counts ( fig3 e ) per 20 × field are presented as mean determinants (± sem ) of 6 - 8 serial sections in each group . the results again clearly demonstrate that the truncated pdgf - c homodimer exhibits marked angiogenic activity in vivo . as can be seen in fig3 d , truncated pdgf - c homodimer ( pdgf - cc ) is able to induce angiogenesis in the mouse cornea similar to other dimeric isoforms of pdgfs including pdgf - aa , pdgf - ab , and pdgf - bb . homodimers of pdgf - bb ( fig3 c ) and pdgf - cc ( fig3 d ), and the heterodimer pdgf - ab ( fig3 b ) induced a similar angiogenic pattern in the mouse cornea . the measured vessel length ( fig3 e ), clock hours ( fig3 f ), and area of neovascularization ( fig3 g ) stimulated by the same amount of these three isoforms were indistinguishable from each other . consistent with the area of vascularization , the immunohistological studies with the anti - cd31 antibody revealed that microvessel densities induced by pdgf - ab , pdgf - bb and pdgf - cc were virtually identical ( fig3 b - e ). in contrast , the vessel length ( fig3 e ) vessel clock hours ( fig3 f ), vascular area ( fig3 g ) and vessel density ( fig3 a and 33e ) stimulated by pdgf - aa were significantly less than those induced by pdgf - ab , pdgf - bb or pdgf - cc ( fig3 and 33 ). all four isoforms of the pdgfs stimulated blood vessels that were dilated ( fig3 a - 32 d ). the test results show that although pdgf - aa also induces angiogenesis in vivo , it does so to a lesser extent than pdgf - cc . it also has been shown that pdgf - aa lacks the ability to directly induce endothelial cell proliferation , migration , and tube formation in vitro ( smits et al ., growth factors 1989 2 1 - 8 ); marx et al ., j clin invest 1994 93 131 - 9 ); koyama et al ., j cell physiol 1994 158 1 - 6 ); sato et al ., am j pathol 1993 142 1119 - 30 ); plate et al ., lab invest 1992 67 529 - 34 ). because pdgf - cc , like pdgf - aa , only activates the pdgfr - a receptor , the different angiogenic activity of pdgf - cc in vivo must be regarded as unexpected . in light of the foregoing test results , which demonstrate the in vivo angiogenesis inducing activity of pdgf - cc , treatments with pdgf - cc alone , or in combination with other angiogenic factors such as vegf and fgf - 2 , provides an attractive approach for therapeutic angiogenesis of ischemic heart and limb disorders . 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 , 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 53 - 64 ; 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 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