Patent Application: US-88486697-A

Abstract:
hemodynamic forces play a key role in inducing 2 , theroscler - osis - implicated gene in vascular endothelial cells . to ellcitate the signal transduction pathway leading to such gene expression , the effects of fluid shearing on the activities of upstream signaling molecules is reported here . fluid shearing induced a transient and rapid activation of p21 ras and preferentially activated c - jun nh 2 terminal kinases over extracellular signal - regulated kinases . co - transfection of rasni7 , a dominant negative mutant of ha - ras , attenuated the shear - activated jnk and luciferase reporters driven by tpa - responsive elements . jnik and mekk , the respective catalytically inactive mutants of jnki and mekk , also partially inhibited the shear - induced luciferase reporters . in contrast , raf301 , erk , and erk , the dominant negative mutants of raf - 1 , erk - 1 , and erk - 2 , respectively , had little effects on the activities of these reporters . the activation of jnk was also correlated with an increased c - jun transcriptional activity , which was attenuated by a negative mutant of son of sevenless . thus , mechanical stimulation exerted by fluid shearing activates , primarily the ras - mekk - jnk pathway in inducing endothelial gene expression .

Description:
following long - standing patent law convention , the terms “ a ” and “ an ” mean “ one or more ” when used in this application , including the claims . several of the above sequences and sequences employed throughout the present specification include reference to particular amino acids . as will be recognized by those of skill in the art , each of the amino acids may be encoded by several different nucleic acid triplet codons . these various nucleic acid triplet codons are noted in table 2 . in some of the nucleic acid sequences , the designation “ n ” is used , and is intended to include a number of different nucleotides that encode a designated group or particular amino acid . the table 2 identifies the several different nucleotide triplet codons that encode a particular amino acid , and may be used in the selection of particular nucleic acid sequences encoding for an identified amino acid within the various nucleic acid sequences in the present disclosure . the invention also provides nucleic acid molecules of the substitution proteins and peptides , and their precursor molecules . in some embodiments , the nucleic acid molecules comprise at least a 10 or 20 nucleotide segment of defined nucleic acid sequences , such as the complement of those set forth at seq id no : 2 , the molecule being capable of hybridizing to the nucleic acid sequence at seq id no : 2 under hybridization stringency conditions standard for hybridization fidelity and stability . these respective molecules may be further defined as comprising a nucleic acid sequence substantially free of nucleic acid sequences that do not encode a protein or peptide that competes with native ras for biological activities of the native ras . in other embodiments , the nucleic molecules of the invention are defined as having a sequence comprising at least a 10 , 20 , 25 , 30 , 40 , 50 , 60 , 70 , 75 , 80 , 90 , 100 , 140 , 150 , 160 , 40 , 450 , 480 , 500 or 510 nucleotide segment of a substituted ras peptide , such as that defined by the n - terminal fragment of substituted sequence for ras at seq id no : 2 . this molecule is further defined as being capable of hybridizing to the complementary sequence of the nucleic acid sequence of seq id no : 2 under relatively stringent hybridization conditions standard for hybridization fidelity and stability . the cdna encoding a substitution mutant of ras peptide having a substituted amino acid residue at position 17 is a further embodiment of the invention . the isolated dna molecules of the invention also may be described as a molecule selected from the group consisting of : ( a ) cdna encoding a biologically active substitution ras protein having a nucleotide sequence derived from the coding region of the sequence at seq id no : 2 , 8 , 9 , 10 or 11 a dna capable of hybridizing to the complementary cdna of ( a ) under moderate conditions of stringency and ( c ) a dna which is degenerate as a result of the genetic code to the dna defined in ( a ) or ( b ) and which encodes a biologically active substitution mutant thereof capable of binding to or complementary binding with ras . in another embodiment , an isolated dna molecule consisting essentially of a nucleotide sequence selected from the group consisting of a nucleotide sequence which encodes an antigenic fragment of said substituted ras protein that includes the substituted amino acid , and a nucleotide sequence which hybridizes to the nucleotide sequence encoding said mutant , is provided . in still other embodiments , the invention concerns isolated dna segments and recombinant vectors that encode a protein or biologically active peptide fragment thereof that includes with its amino acid sequence an amino acid sequence essentially as set forth in seq id no : 4 . such references are also made in relation to the description of particular nucleotide sequences , such as those of seq id no : 8 . the term “ a sequence essentially set forth in seq id no :” means that the sequence substantially corresponds to a portion of the identified seq id no :, whether it be referencing an amino acid or nucleic acid sequence identifier , and has relatively few amino acids or nucleotide bases , as the case may be , which are not identical to , or biologically functional equivalent of , the amino acids or the nucleotides of the designated seq id no : bovine aortic endothelial cells ( baec ) prior to passage 10 were used in all the studies . the cells were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) supplemented with 10 % fetal bovine serum . all cell cultures were kept in a humidified 5 % c0 2 - 95 % air incubator at 37 ° c . baec were cultured on 38 mm × 76 mm slides to confluence , and the slides were then assembled into a rectangular flow channel with a height of 270 μm , through which the medium flows . the system was tightly sealed by using a silicon gasket and a vacuum line . a surface area of 14 cm 2 on the baec - seeded slide , confined by the gasket , was exposed to the applied fluid shearing , which was generated by circulating the tissue culture medium through a hydrostatic pump connected to upper and lower reservoirs ( 15 ). the ph of the system was kept constant by gassing with 95 %. air - 5 % c0 2 , and the temperature was maintained at 37 ° c . by immersing the flow system in a water bath . the shear stress , determined by the flow rate perfusing the channel and the channel thickness , was 12 dynes / cm 2 ( 1 dyne = 10 − 5 newton ) which is relevant to the physiological range in the human major arteries and has been found to induce the expression of many ie genes in vitro ( 22 , 40 ). the duration of the applied fluid shearing was 8 hr in the gene regulation experiments and varied from 1 to 60 min in the signal transduction experiments . static control experiments were performed on baec on slides not exposed to fluid shearing . the assays were performed according to the procedures described previously by downward et al . ( 12 ) with minor modifications . baec cultured on a glass slide were labeled with 0 . 5 mci / ml [ 32 p ] orthophosphate ( icn radiochemicals ) for 6 hr in a phosphate - free medium . after labeling , the cells were subjected to fluid shearing or kept as static controls . cell extracts were then prepared afterwards by lysing the baec in a buffer containing 50 mm tris - hc1 , ph 7 . 5 , 0 . 5 %- np - 40 , 0 . 15 m nac1 , 0 . 1 mm na 3 vo 4 , 20 mm mgc1 2 , 0 . 5 % trotpm x - 100 , 1 μg / ml leupeptin , 1 mm pmsf , 2 mm dtt , and 2 mm β - glycerolphosphate . ras proteins were immunoprecipitated with rat anti - p21 ras mab ( santa cruz ). the bound guanine nucleotides were separated from the precipitated protein complexes by using a buffer containing 20 mm tris - hcl , ph 7 . 5 , 20 mm edta , 2 mm dtt , 2 % sds , and 2 mm gtp . the eluted nucleotides were separated by thin layer chromatography using pei - cellulose plates with 0 . 75 m k 2 hp0 4 , ph 3 . 4 . the gdp and gtp contents were assessed by autoradiography . five micrograms of anti . mapk / protein a - agarose ( ubs ), in a buffer containing 20 mm hepes ( ph 7 . 7 ), 75 mm nacl , 2 . 5 mm - mgcl 2 , 0 . 1 mm edta , 0 . 05 % triton x - 100 , 0 . 5 mm dtt , 20 mm β - glycerolphosphate , 0 . 1 mm na 3 vo 4 , 2 μg / ml leupeptin , and 100 μg / ml pmsf , were added to the cell lysate to immunoprecipitate erk . the suspension was mixed in 4 ° c . for 4 hr and centrifuged . the pelleted beads were washed in phosphate buffer saline containing 0 . 1 % triton x - 100 , followed by resuspension in 30 μl of a kinase buffer which contained 20 mm hepes ( ph 7 . 6 ), 20 mm mgcl 2 20 mm β - glycerolphosphate , 20 mm p - nitrophenyl phosphate , 0 . 1 mm n 3 vo 4 , 2 mm dit , 20 μm atp , 5 μg myelin basic protein ( mbp ), and 5 μci [ γ 32 p ] atp . after incubation at 30 ° c . for 20 min , the kinase reaction was terminated by washing with hepes binding buffer . the phosphorylated proteins were eluted in 30 μl of 2 × laemelli sample buffer and resolved on 10 % sds - polyacrylamide gel , followed by autoradiography . the procedures for jnk activity assay were the same as for erk , except that 1 μg agarose - bound glutathione - s - transferase ( gst )- c - jun ( 1 - 223 ) ( 19 ) and 5 μci [ γ 32 p ] atp were added directly to the cell lysate for kinase reaction . in some of the experiments , plasmid encoding ha - jnk was transfected into baec and the exogenous epitope - tagged was immunoprecipitated with a mouse anti - ha mab ( boehringer mannheim ). the following procedures to assay the activity of the jnk were the same as described above . erk ( k52r ) and erk ( k71r ) were gifts from dr . melanie cobb at university of texas . ha - jnk , ga14 - c - jun ( 1 - 223 ), ga14 - c - jun ( 1 - 223 , ala63 / 73 ), mekk ( k - m ), raf301 , rasni7 , msos1 , and δmsos1 were described previously ( 10 , 14 , 24 , 32 , 34 , 39 ). to construct jnk ( k - r ), jnk 1 was mutated in pbluescript by pcr to introduce a ncoi site at its first atg codon and a point mutation at codon 55 which replaced the lys - 55 with an arg . the mutations were confirmed by dna sequencing . the mutated jnk was then subcloned into expression vector srα3ha ( 9 ) at ncoi and bgiii sites to create jnk ( k - r ). expression plasmids encoding the wild - type , active , or dominant negative mutants of p21 ras , mekk , or jnk were co - transfected with either 4 × tre - p1 - luc or mcp1 - luc - 540 into baec at 70 % confluence by using the transient transfection protocols . 4 × tre - p1 - luc is a construct in which the rat ras promoter conjugated to luciferase reporter is driven by four copies of the tre consensus sequence , and mcp1 - luc - 540 is a construct in which the luciferase reporter is driven by the 540 - bp mcp - 1 promoter ( 41 ). the psv - β - galactosidase plasmid , which contains a β - galactosidase ( β gal ) gene driven by sv40 promoter and enhancer , was also included in the co - transfection to monitor the transfection efficiency . after incubation for 6 hr , the cells were washed with pbs and incubated with fresh media for another 24 - 48 hr to reach confluence . the cells in the tissue culture flasks were then seeded on glass slides and used either for fluid shearing experiments or as static controls . the luciferase reporter activities of the various experiments normalized for transfection efficiency were used to assess the suppressing effects of the various negative mutants on the shear - induced transcription activation mediated by ap - 1 / tre . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . to investigate whether fluid shearing leads to an activation of p21 ras in ecs , i . e ., an increased ratio of p21 ras gtp to p21 ras gdp , confluent monolayers of the 32 p - labeled baec were subjected to a fluid shearing of 12 dynes / cm 2 for various time periods , and the cells were lysed and subjected to guanine nucleotide binding assays . in the static controls , p21 ras was exclusively in its gdp - bound inactive form ( fig1 ). after shearing for 1 min ., the ratio of p21 ras gtp / p21 ras gdp increased markedly . densitometric analysis indicated that 17 ± 4 % of all the guanine nucleotides bound to p21 ras was gtp bound . this gtp - bound active from gradually returned to the gdp - bound form afterwards . by 5 min after the beginning of shearing , the gtp bound form decreased to 7 ± 3 %. by 10 min , all p21 ras became inactive , as in the static controls . thus , fluid shearing , like other extracellular stimuli such as mitogens , cytokines , osmotic shock , and uv irradiation , induces a transient activation of p21 ras . by using transient transfection assays , the present inventors show that the luciferase reporter driven by tre ( i . e ., 4 × tre - p1 - luc and mcp1 - luc - 540 ) can be induced by fluid shearing . transactivation asssays using c - jun or c - jun / c - fos expression plasmids also induced these tre - driven constructs ( 41 ), indicating that the transcription factor that mediates such activation is ap - 1 . to investigate whether the ras is upstream to the shear - induced activation of ap - 1 / tre , we examined the effects of rasn17 on the induction of 4 × tre - p1 - luc and mcpi - luc - 540 . rasn17 is a dominant negative mutant of ras in which ser - 17 in the wild - type has been replaced by asn , so that the affinity to gtp is dramatically reduced ( 14 ). as shown in fig2 , in the plasmid control experiments , fluid shearing caused 23 and 6 . 5 folds of induction ( the luciferase activates in the sheared cells compared to those in the static controls ) for 4 × tre - p1 - luc and mcpi - luc - 540 , respectively . the present inventors also transfected expression plasmid encoding rasl61 , the active form of p21 ras in which the gln - 61 in the wild - type has been replaced by leu , into baec . the expression of rasl61 increased the basal level expression for both 4 × tre - p1 - luc and mcpi - luc - 540 . however , the induction by fluid shearing was not affected . in contrast , the co - transfection of rasn17 with either 4 × tre - p1 - luc or mcpi - luc - 40 into baec significantly decreased the shear - induced luciferase reporter activities . these results , together with those presented in fig1 suggest that a functional p21 ras is required for the ap - 1 / tre - mediated gene expression in response to fluid shearing . in ecs , erk - 1 and - 2 are shown to be phosphorylated by fluid shearing ( 41 ). to investigate whether the phosphorylation of erk led to their increased kinase activities , baec were subjected to shearing at 12 dynes / cm 2 for various lengths of time . as shown in fig3 a , such a mechanical stimulation induced a rapid activation of erk to cause mbp phosphorylation , which peaked at 10 min and decreased afterwards . densitometric analysis indicated that the peak activity was 1 . 8 folds of that in the static controls . to investigate whether fluid shearing activates jnk as it does for erk , agarose - bound gst - c - jun , a fusion protein containing gst and the n - terminal moiety ( 1 - 223 ) of c - jun , and [ γ - 32 p ] atp were added to the cell lysate for jnk in baec to cause c - jun phosphorylation , which peaked at 30 min and decreased afterwards . after the cells had been exposed to the shearing for 60 min , the jnk activities returned to a level lower than that in the static controls . the jnk activity peaked , at 30 min and decreased afterwards . after the cells had been exposed to the shearing for 60 min , the jnk activities returned to a level lower than that in the static controls . the peak jnk activity , at 30 min , determined by densitometry , was 10 . 5 folds of that in the static cells . kinase assays on static cells incubated with fresh media or with conditioned media collected from sheared cells showed no difference in kinase activities from the static controls ( data not shown ). these results indicate that the activation of cytoplasmic kinases in the sheared cells was attributable to the action of mechanical force rather than by the media supplements or by the metabolites released from the cells during shearing . furthermore , fluid shearing of baec activates jnk to a greater extent and for a longer duration than erk . p21 ras and mekk are upstream to the shear - activated jnk whether p21 ras is upstream to such shear - activated jnk was further investigated . kinase assays shown in fig4 indicate that the exogenous epitope - tagged ha - jnk was also activated by fluid shearing in the transfected baec . the co - transfection of rasn17 inhibited such shear - induced kinase activity of ha - jnk as manifested by the phosphorylation of its substrate gst - c - jun ( fig4 a ). it has been shown that the expression of ras activates mekk ( 27 ) and that the induction of mekk stimulates jnk ( 48 ). co - transfection of a catalytically inactive enzyme mekk ( k - m ), in which the lys - 432 had been replaced by a met ( 32 ), with ha - jnk also reduced the shear - induced jnk activity ( fig4 a ). these results indicate that fluid shearing activates a ras - mekk - jnk pathway in the vascular ec &# 39 ; s . to further examine whether mekk mediates the shear - induced reporter driven by ap - 1 / tre , co - transfection of mekk ( k - m ) with either 4 × tre - p1 - luc - 540 was done . in addition , jnk ( k - r ), a kinase deficient jnk1 , in which the lys - 52 in the wild - type was replaced by an arg , was constructed . if the ras - mekk - jnk pathway is upstream to the ap - 1 / tre , the use of either mekk ( k - m ) or jnk ( k - r ) to block the functions of the wild - types should attenuate the shear - induced ap - 1 / tre . fig5 a indicates that the co - transfection of expression plasmids encoding mekk ( k - m ) or jnk ) k - r ) did attenuate the shear - induced 4 × tre - p1 - luc from 21 . 5 folds to 12 . 5 or 4 folds , respectively . co - transfection of these catalytically inactive mutants with mcpi - luc - 540 also reduced the shear induced luciferase reporter activities ( fig5 b ). in contrast , co - transfection of the expression plasmids encoding the wild - type mekk or jnk did not affect the shear - induced 4 × tre - p1 - luc or mcpi - luc - 540 . thus , mekk and jnk are upstream to the ap - 1 / tre mediated gene expression in response to shear stress . raf - 301 , erk ( 52r ), and erk ( 71r ) have little effects on the shear - activated ap - 1 / tre in response to the stimulations by growth factors or phorbol ester , the ras - erk pathway is activated ( 11 , 46 ), leading to the activation of ap - 1 / tre ( 16 ). raf - 1contributes directly to erk activation in this pathway , but not to jnk activation ( 23 ). mechanical shearing has a less potent effect on erk than on jnk ( fig3 ), which seams to indicate that the ras - raf - 1 - erk pathway is less important for the downstream gene expression . to test the role played by he ras - raf - 1 - erk pathway in the shear - induced activation of ap - 1 / tre , we used the dominant negative mutants of raf - 1 and erk to block this pathway and examined the ap - 1 / tre - mediated reporter activities in response to mechanical stimulation . raf301 is a dominant negative mutant of raf - 1 in which the lys - 375 in the wild - type has been replaced by trp ( 24 ). erk ( k71r ) and erk ( k52r ) are the dominant mutants of erk1 and erk2 , in which the respective lys71 and lys - 52 in the wild types as been replaced by arg ( 36 ). fig6 shows that co - transfection of ra301 had little effect on the shear - induced 4 × tre - p1 - luc reporter activity . similarly , neither erk ( 71r ), erk ( 52r ), nor a combination of these two erk dominant negative mutants attenuated the reporter activity in response to fluid shearing . experiments using mcp1 - luc - 540 also showed that none of these negative mutants was able to affect the shear - induced luciferase activity ( data not shown ). thus , the ras - raf - 1 - erk pathway is not essential for the shear - induced activation of ap - 1 / tre . it seems that the induction of 4 × tre - p1 - luc and mcpi - luc - 540 by fluid shearing results from an up - regulated c - jun , which is activated by the ras - mekk - jnk pathway to test whether fluid shearing increases the transcriptional activity of c - jun , plasmid ga14 - c - jun encoding the fusion protein of ga14 dna binding domain and c - jun activation domain ( 1 - 223 ) were co - transfected with 4 × ga1 - luc , a chimeric construct consisting of he ga14 - binding sequence and the luciferase reporter , into baec . compared to the static controls , fluid shearing increased the luciferase activity by more than 4 folds in the sheared cells ( fig7 ), indicating an increased c - jun transcriptional activity . in contrast , the plasmid encoding the mutated ga14 - jun , in which the phosphorylation sites ser - 63 and - 73 had been replaced by ala , showed a marked reduction in response to fluid shearing . furthermore , co - expression of rasn17 or mekk ( k - m ) also attenuated such shear - induced transcriptional activity . thus , the fluid - shearing induced activation of ap - 1 is at least in part due to an increased c - jun transcriptional activity , which is in turn activated through the phosphorylation of ser - 63 and - 73 by the ras - mekk - jnk pathway . son of sevenless ( sos ) is a guanine nucleotide exchange factor that activates p21 ras by converting the gdp - bound inactive state to the gtp - bound active state ( 4 , 13 ). to explore whether sos is a upstream molecule regulating the shear - activated ras signaling , expression plasmids encoding δmsos1 , a dominant negative mutant of muse sos1 in which the guanine nucleotide exchange domain has been deleted ( 39 ), were co - transfected with ga14 - c - jun and 4 × gal - luc into baec . the transfected cells were then subjected to fluid shearing / luciferase assays . as shown in fig7 b , δmsos1 attenuated the shear - induced c - jun transcriptional activity . the inducibiliy by fluid shearing in cells transfected with msos1 , the wild - type mouse sos1 , was comparable to that in cells transfected with empty vectors . a recombinant adenovirus , adrasn17 , was constructed by cotransfecting 293 embryonic kidney cell line with paccmvplpa ( 8 . 8 kb ), a shuttle vector containing rasn17 cdna , and pjm17 , a vector carrying a dl309 adenovirus 5 genome ( ad5 ). after the homologous recombination between paccmbplpa and pjm17 in 293 cells , the adenovirus e1 region responsible for viral replication was substituted by rasn17 expression cassette , resulting in replication - deficient viruses . southern blot assay was performed to identify the viruses containing the rasn17 dna . the of this preparation was determined by optical density measurement . rasn17 inhibits the ap - 1 / tre - mediated transactivation in response to serum or pdgf the cloned rasn17 adenovirus shuttle vector and a luciferase reporter driven by tpa - responsive elements ( 4 × tre - pl - luc ) were co - transfected into bovine aortic endothelial cells ( baec ). the transfected cells in serum - free medium were treated with either 15 % serum or 10 ng / ml pdgf for 24 hr , and this was followed by luciferase activity assays . fig2 shows that the induction of luciferase activity by serum or pdgf in the control cells is significantly attenuated by the co - transfection of rasn17 . these results indicate that the ap - 1 / tre - mediated gene expression ( e . g ., mcp - 1 ) induced by mitogenic stimuli in vascular endothelium can be blocked by rasn 17 . a recombinant adenovirus , adrasn17 , was assembled by transfecting 293 embryonic kidney cell line with paccmv p l p a ( 8 . 8 kb ), a shuttle vector containing rasn17 cdna , and pjm17 , a vector carrying a dl309 adenovirus 5 genome ( ad5 ). the pjm17 contains the full length ad5 genome ( 36 kb ) with the interruption of a 4 . 3 kb unrelated dna fragment at position 3 . 7 map units , thereby exceeding the adenoviral packaging limit . after the homologous recombination between paccmb p l p a and pjm17 in 293 cells , the adenovirus e1 region responsible for viral replication was substituted by the rasn17 expression cassette , resulting in replication - deficient viruses . putative viral clone were plaque purified , propagated , isolated , and the tier was determined according to the established procedures . southern blot was performed to verify the insertion of rasn17 dna into viruses . the control adenoviral vector containing a lacz gene expression cassette ( adlacz ) was constructed by similar procedures . adlacz was obtained from 293 cells co - transfected with pjm17 and pxcjl 1 / cmv / n1s - lacz , a derivative of pxcjl . 1 that carries an expression cassette in which the human cytomegalovirus ie promoter encoding the sv40 large t - antigen nuclear - localization signal was fused to the e . coli lacz reporter gene . a primer with sequence 5 ′- ttgtggacgaatacgacc - 3 ′ corresponding to the 5 ′ end of p21ras cdna was used to sequence the rasn17 ( seq id no : 16 ) cloned into the adenoviral vector pjm17 . the partial sequence of the cloned rasn17 is as follows : ad - rasn17 inhibits the serum and pdgf - induced mitotic responses in pig smooth muscle cells by introducing a reporter gene lacz , it was found that the transfection efficiency of dna into porcine smooth muscle cells by liposome is poor . only approximately 5 % of cells can be transfected , as demonstrated by x - gal staining in fig1 a . in order to test dna transfection efficiency mediated by adenovirus , an adenovirus containing lacz gene , ad - lacz , was used to determine the transfection efficiency . as shown in fig1 b , the majority of the smooth muscle cells (& gt ; 95 %) are x - gal staining positive , indicating the successful injection of these cells by ad - lacz . thus , adenoviral vector is superior to liposomes as a delivery system to transfer therapeutic genes into vascular cells . an adenovirus - based system to deliver the negative mutants in the ras - mediated signaling pathway to vascular cells was developed . these molecules , which include , but are not limited to , negative mutants of son of sevenless ( sos ), mekk , jnkk , jnk , raf and erk , have been previously shown to attenuate the downstream gene expression . this development is aiming at blocking the ras pathway in a comprehensive manner so that the expression of genes involved in atherogenesis and restenosis can be effectively abolished . rasn17 inhibits the induction of ap - 1 / tre by serum or pdgf in endothelial cells the cloned rasn17 adenovirus shuttle vector and a luciferase reporter driven by tpa - responsive elements ( 4 × tre - p1 - luc ) were co - transfected into bovine aortic endothelial cells ( baec ). each group of cells were cultured in serum - free medium for 24 hours followed by addition of ether 15 % serum or 10 ng / ml platelet derived growth factor ( pdgf ) for another 24 hour incubation , followed by luciferase activity assays . fig2 shows that the induction of luciferase activity by serum or pdgf in the control cells is significantly attenuated by the co - transfection of rasn17 . these results indicate that the ap - 1tre - mediated gene expression ( e . g ., mcp - 1 ) induced by mitogenic stimuli in vascular endothelium can be blocked by rasn17 . use of rasn17 to attenuate vascular smooth muscle proliferation in vitro the present example is provided because the proliferate response of vsmc to high - serum or pdgf culture conditions in vitro resembles heir hyperplastic response to balloon injury in vivo . 15 % serum or 10 ng / ml pdgf was applied to the serum starved porcine vsmc after transfection with a replication - deficient virus carrying the rasn17 gene ( ad - rasn17 ). in controls , replication - deficient virus carrying the lacz gene ( ad - lacz ) was used . the 3 h - thymidine incorporation assay was performed to assess cell proliferation . in vitro , vsmc stimulated with serum and pdgf showed proliferate response in the ad - lacz control group , but this was inhibited in the ad - rasn17 transfected group . psmcs were seeded on 96 - well plates until 50 % confluence . the cells were then infected with the replication - deficient adenovirus adlacz or adrasn17 in 1 × 10 6 , 1 × 10 7 , or 1 × 10 8 plaque - forming units per milliliter ( pfu / ml ). after 24 - hr infection , the infected cells were starved in dmem containing 0 . 5 % serum for 24 hr followed by stimulation with 15 % serum or 10 ng / ml pdgf . the cells were pulse - labeled for 4 hr in growth media containing 2 . 5 μc / ml methyl - 3 h thymidine ( amersham life science ). the cells were trypsinized and collected on glass fiber filter papers . the filter papers were collected in polypropylene vials , mixed with 5 ml scintillate per sample for 12 hr , and counted in a beta scintillation counter . serum - starved psmcs infected with a replication - deficient virus carrying the rasn17 gene ( ad - rasn17 ) or the control replication - deficient virus carrying the lacz gene ( ad - lacz ) were subjected to stimulation with 15 % serum or 10 ng / nl pdgf . as shown in fig9 a and 9b , 3 h - thymidine incorporation assay showed that cell proliferation increased by 48 times in the serum - stimulated psmcs and 15 times in the pdgf - stimulated psmcs infected with ad - lacz ( 10 8 pfu / ml ) as controls . in contrast , the serum - and pdgf - stimulated proliferation was reduced drastically for psmcs infected with ad - rasn17 at 10 8 pfu / ml . these results indicate that expression of rasn17 in smc can significantly attenuate the growth of the cells . use of ras n17 in gene therapy to prevent artery restenosis in vivo percutaneous transluminal coronary angioplasty ( ptca ) has been extensively used as a clinical approach to treat coronary heart disease . however , restenosis occurs at the site of angioplasty in approximately one third of the patents within 6 months after ptca . an important factor in restenosis is that the abrasive actions on the vessel wall during the ptca procedure denude the endothelial cells and traumatize the vessel media , leading to the inflammatory and proliferative responses of smooth muscle cells to cause restenosis . our in vitro studies on culture pig smooth muscle cells showed that the introduction of rasn17 , a dominant negative mutant of p21ras , into those cells inhibited the expression of genes that lead to cell proliferation in response to several mitogens ( e . g ., pdgf ) and hemodynamic force ( e . g . shear stress ). these data led us to perform animal experiments to test the in vivo efficacy of negative mutants in the ras pathway in preventing restenosis after ptca . the aim is to provide an effective method for the reduction of the high incidence of restenosis after angioplasty and other surgical interventions in patients with coronary heart disease . our results show that the restenosis of rat common carotid arteries after balloon injury is inhibited by local treatment with recombinant adenovirous carrying rasn17 . the rats were anesthetized with ketamine ( 100 mg / kg body weight , i . p .) and xylazine ( 10 mg / kg body weight , i . p .). under sterile conditions , a neck incision was made and the left carotid artery was exposed . the common carotid artery ( cca ) was clamped at 2 cm proximal to the bifurcation , and the internal carotid artery ( ica ) was also clamped at its proximal position . the external carotid artery ( eca ) was ligated at 1 cm distal to the bifurcation and a small arteriotomy was made just proximal to the ligation site for the insertion of a balloon catheter into the cca . vascular injury was achieved by inflating the balloon at 1 . 8 atm , and then sliding the balloon catheter back and forth three times . after the deflation of the balloon and the withdrawal of the catheter , a vascular clamp was placed at 1 cm proximal to the bifurcation , and adenovirus ( 50 μl , 10 9 pfu / ml ) was injected through the arteriotomy into the segment distal to the clamp . after 15 - min incubation , the virus was removed , and the clamp was removed to restore blood flow . the neck incision was closed , and the rat was allowed to recover with normal husbandry procedures for two weeks . then , the animal was sacrificed with an overdose of anesthesia and perfused with pbs and 4 % para - formaldehyde phosphate buffer at 100 mmhg for 10 - 15 min . the cca was removed and fixed overnight for histological staining with hemtaoxylin - eosin . the balloon procedures in the common carotid arteries of ten rats was performed with ad - rasn17 or ad - lacz injected into the distal segment of the vessel . ad - rasn17 was injected in five of the animals as the experimental group , and ad - lacz in the other five as controls . histological examination was made to determine the intimal / media cross - sectional area ratio ( i / m ratio ) for the assessment of restenosis . in the control group , two of the five animals developed restenosis in both the proximal and distal segments ( fig1 b ), and the i / m ratio was 1 . 99 ± 0 . 23 . in the experimental group , restenosis developed in two of the five animals , but only in the proximal , untreated segment of their vessels ; the i / m ratio there was not significantly different from that in the control group . the distal segments of these experimental animals , where ad - rasn17 injection was made , showed little evidence of restenosis ( fig1 c ), and the i / m ratio ( 0 . 95 ± 1 . 15 ) was markedly lower than that in the proximal segment of the same animals or the segments in the control group . these results suggest that ad - rasn17 is a potential therapeautical gene for the prevention of post angioplasty restenosis . the expression of rasn17 in porcine muscle cells in vitro significantly attenuates cell proliferation in response to growth stimulations . the administration of rasn17 into rat common carotid arteries markedly reduced the restenosis of the vessel after balloon injury in vivo . therefore , ad - rasn17 is a potential therapeutical gene for the prevention of post angioplasty restenosis . use of rasn17 derivatives as therapeutic gene in the prevention of restenosis a partial sequence of rasn17 containing the mutated gtp binding site is postulated to be sufficient to be functional in inhibiting the proliferation of smooth muscle cells after ptca . this truncated gene contains the 80 amino acids at the nh 2 - terminal of rasn17 with the sequence shown below ( seq id no : 25 ): in addition , several mutants , e . g ., rasa17 , rasg17 , and rask17 in which ser - 17 in the wild - type is replaced by ala , gly , or lys , respectfully , will be constructed ( see table below for coding sequence ). these mutants will be tested for their therapeutic effects in prevention of restenosis after ptca . balloon procedures in femoral arteries of two pigs have been performed . restenosis was found in both of the animals 40 days after the procedures . cross - sectional histology of the arterial specimen showed that the injured vessels were occluded ( compare the normal artery in the left panel to the injured one in the right panel of fig1 ). experiments with ad - rasn17 injection are currently conducted to investigate the inhibitory effects on restenosis . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . 1 . angel , p ., and m . karin . “ the role of jun , fos and the ap - 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and phorbol este - induced tyrosine phosphorylation of map kinases ,” cell , 68 : 1031 - 1040 , 1992 . 48 . wang , et al ., “ mechanotransduction across the cell surface and through the cytoskeleton ,” science , 260 : 1124 - 1127 , 1993 . 49 . yan , et al ., “ activation of stress - activated protein kinase by mekk1 phosphorylation of its activator sek1 , ” nature , 372 : 798 - 800 , 1994 . atg acg gaa tat aag ctg gtg gtg gtg ggc gcc ggc ggt gtg ggc aaa 48 aat gcg ctg acc atc cag ctg atc cag aac cat ttt gtg gac gaa tac 96 asn ala leu thr ile gln leu ile gln asn his phe val asp glu tyr gac ccc act ata gag gat tcc tac cgg aag cag gtg gtc att gat ggg 144 asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly gag acg tgc ctg ttg gac atc ctg gat acc gcc ggc ctg gag gag tac 192 agc gcc atg cgg gac cag tca atg cgc acc ggg gag ggc ttc ctg tgt 240 ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys gtg ttt gcc atc aac aac acc aag tct ttt gag gac atc cac cag tac 288 val phe ala ile asn asn thr lys ser phe glu asp ile his gln tyr agg gag cag atc aaa cgg gtg aag gac tcg gat gac gtg ccc atg gtg 336 ctg gtg ggg aac aag tgt gac ctg gct gca cgc act gtg gaa tct cgg 384 leu val gly asn lys cys asp leu ala ala arg thr val glu ser arg cag gct cag gac ctc gcc cga agc tac ggc atc ccc tac atc gag acc 432 gln ala gln asp leu ala arg ser tyr gly ile pro tyr ile glu thr tcg gcc aag acc cgg cag gga gtg gag gat gcc ttc tac acg ttg gtg 480 ser ala lys thr arg gln gly val glu asp ala phe tyr thr leu val cgt gag atc cgg cag cac aag ctg cgg aag ctg aac cct cct gat gag 528 agt ggc ccc ggc tgc atg agc tgc aag tgt gtg ctc tcc tga 570 asn ala leu thr ile gln leu ile gln asn his phe val asp glu tyr asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys val phe ala ile asn asn thr lys ser phe glu asp ile his gln tyr leu val gly asn lys cys asp leu ala ala arg thr val glu ser arg gln ala gln asp leu ala arg ser tyr gly ile pro tyr ile glu thr ser ala lys thr arg gln gly val glu asp ala phe tyr thr leu val xaa ala leu thr ile gln leu ile gln asn his phe val asp glu tyr asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys xaa ala leu thr ile gln leu ile gln asn his phe val asp glu tyr asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys val phe ala ile asn asn thr lys ser phe glu asp ile his gln tyr leu val gly asn lys cys asp leu ala ala arg thr val glu ser arg gln ala gln asp leu ala arg ser tyr gly ile pro tyr ile glu thr asn ala leu thr ile gln leu ile gln asn his phe val asp glu tyr asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys val phe ala ile asn asn thr lys ser phe glu asp ile his gln tyr leu val gly asn lys cys asp leu ala ala arg thr val glu ser arg gln ala gln asp leu ala arg ser tyr gly ile pro tyr ile glu thr ser ala lys thr arg gln gly val glu asp ala phe tyr thr leu val xaa ala leu thr ile gln leu ile gln asn his phe val asp glu tyr asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys val phe ala ile asn asn thr lys ser phe glu asp ile his gln tyr leu val gly asn lys cys asp leu ala ala arg thr val glu ser arg gln ala gln asp leu ala arg ser tyr gly ile pro tyr ile glu thr ser ala lys thr arg gln gly val glu asp ala phe tyr thr leu val asn ala leu thr ile gln leu ile gln asn his phe val asp glu tyr asp pro thr ile glu asp ser tyr arg lys gln val val ile asp gly ser ala met arg asp gln ser met arg thr gly glu gly phe leu cys