Short-Term Local Delivery of an Inhibitor of RasFarnesyltransferase Prevents Neointima Formation In Vivo

After Porcine Coronary Balloon AngioplastyLorraine M. Work, BSc, PhD; Allan R. McPhaden, BSc, MBChB, MD, MRCPath;

Nigel J. Pyne, BSc, PhD; Susan Pyne, BSc, PhD; Roger M. Wadsworth, BPharm, PhD, DSc;Cherry L. Wainwright, BSc, PhD, FESC

Background—Mitogenic stimuli present at the site of coronary arterial balloon injury contribute to the progression anddevelopment of a restenotic lesion, many signaling through a common pathway involving the small G protein p21ras. Ouraim was to demonstrate in biochemical studies that farnesyl protein transferase inhibitor III (FPTIII) is an inhibitor ofp21ras processing and that when it is given locally in vivo at the site of coronary balloon injury in a porcine model, itcan inhibit neointima formation.

Methods and Results—FPTIII (1 to 25mmol/L) concentration-dependently reduced p21ras levels in porcine coronary arterysmooth muscle cell membranes. FPTIII also prevented p42/p44 MAPK activation and DNA synthesis in response toplatelet-derived growth factor in these cells at a concentration of 25mmol/L. Application of 25mmol/L FPTIII locallyfor 15 minutes to balloon-injured porcine coronary arteries in vivo prevented neointima formation assessed at 4 weeks,reduced proteoglycan deposition, and inhibited adventitial hypertrophy. Coronary arteries from FPTIII-treated pigs hadno deterioration in contraction or in endothelium-dependent relaxation.

Conclusions—The study demonstrates in the pig that short-term local delivery of inhibitors of p21ras-dependent mitogenicsignal transduction prevents restenosis after balloon angioplasty.(Circulation. 2001;104:1538-1543.)

Key Words: neointiman farnesyl transferase inhibitorn arteriesn angioplasty

The main mechanisms thought to underlie restenosisinclude smooth muscle cell (SMC) migration and prolif-

eration, adventitial remodeling, and matrix production.1 Nu-merous growth factors and mitogenic autocoids that couldaccelerate new tissue formation and development of therestenotic lesion have been found at the site of injury.2,3

Signal transduction by these agents involves the mitogen-ac-tivated protein kinase (MAPK) pathway, and p21ras-dependent activation of p42/p44 MAPK has been demon-strated in vivo in a porcine model of balloon injury early afterangioplasty.4 Moreover, the local delivery of H-rasdominantnegative mutant (N17 and L61, S186) plasmid constructs5

and adenovirus-mediated transfer of dominant negativeH-ras6 have both been shown to significantly reduce neoin-tima formation in the rat carotid artery.

Farnesyl transferase (FTase) is an enzyme that catalyzesthe insertion of a farnesyl moiety onto the carboxy terminusof p21ras, this being an essential step in the membraneadherence of the protein before activation of the MAPKpathway. Furthermore, FTase also catalyzes Rho farnesyla-tion, another small-molecular-mass G protein involved in a

separate pathway that plays a significant role in regulatingcell proliferation. Inhibitors of FTase have been under devel-opment for applications in cancer therapy for several years7

because of their ability to prevent excessive cell growth incancer cell lines. Inhibitors of FTase can also inhibit prolif-eration8,9 and migration8 of vascular SMCs in vitro. It was ourhypothesis, therefore, that an inhibitor of FTase could be anappropriate means of decreasing neointima formation afterballoon angioplasty. FTase inhibitors may have an advantageover existing specific gene-targeting therapies or antisenseoligonucleotide therapies, because they can target both Ras-and Rho-dependent signaling.10 In this article, we focused onwhether the FTase inhibitor {(E,E)-2-[2-oxo-2-[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]aminoethyl] phosphonicacid, (2,2-dimethyl-1-oxopropoxy) methyl ester sodium}(farnesyl protein transferase inhibitor III, FPTIII)11 acts onRas-dependent activation of p42/p44 MAPK and blockssmooth muscle proliferation. These experiments enabled us toestablish an effective concentration of FPTIII for subsequentlocal delivery after angioplasty in vivo to determine its effectson neointima formation and vascular function.

Received March 27, 2001; revision received June 22, 2001; accepted June 26, 2001.From the Department of Physiology and Pharmacology, University of Strathclyde, and Department of Pathology, Glasgow Royal Infirmary (A.R.M.),

Glasgow, Scotland, UK. Dr Work is now at the Department of Medicine and Therapeutics, Western Infirmary, Glasgow.Correspondence to Dr C.L. Wainwright, Department of Physiology and Pharmacology, University of Strathclyde, Strathclyde Institute for Biomedical

Sciences, 27 Taylor St, Glasgow G4 0NR, Scotland, UK. E-mail c.l.wainwright@strath.ac.uk© 2001 American Heart Association, Inc.

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MethodsBiochemical StudiesPorcine vascular SMCs (PVSMCs) were obtained by explantation of5-mm coronary artery rings into 5 mL of medium (1:1 Ham’s F12and Waymouth’s MB 752/1) containing 15% FCS and 1% penicillin-streptomycin (5000 IU/mL). Cells were used between passages 3 and5. PVSMCs were incubated in medium containing 1% FCS for 24hours to synchronize the cell cycle to G0. FPTIII (1 to 25mmol/L)was added for 18 to 24 hours before stimulation with platelet-derivedgrowth factor (PDGF) (10 ng/mL), endothelin 1 (ET-1) (100 nmol/L), phorbol 12-myristate 13-acetate (PMA) (1mmol/L), or bradyki-nin (200 nmol/L). Cell lysates were analyzed for activation ofp42/p44 MAPK by an in vitro kinase assay or by an electrophoreticmobility shift assay.12 Similarly, membrane-associated p21ras wasdetected by Western blotting. Cell proliferation was determined asPDGF-induced [3H]thymidine incorporation in the absence andpresence of 1 to 25mmol/L FPTIII.13 Shift blots of PDGF- andPMA-induced phosphorylation of p42 MAPK were also performedin the absence and presence of 1 to 25mmol/L FPTIII, according toConway et al.12

In Vivo Coronary Balloon AngioplastyAll experimental procedures were performed under the guidelines ofthe UK Home Office Scientific Procedures (1986) Act. Male LargeWhite/Welsh Landrace pigs (n518; 14 to 19.5 kg) were premedi-cated with aspirin (325 mg PO), sedated with azaperone (5 mg/kgIM), and anesthetized with a mixture of O2/halothane/nitrous oxide.An introducer sheath (7F) was inserted into the artery to allow a 6Fguide catheter to be advanced to the coronary ostium. A 3-mmballoon angioplasty catheter was then advanced via the guidecatheter (under fluoroscopic control) to the midpoint between thefirst and second diagonal branches of the left anterior descendingcoronary artery (LAD). After a coronary angiogram to confirm thepositioning of the balloon catheter, the balloon was inflated 3 timesto 10 atm, providing an approximate balloon-to-artery ratio of 2:1. In11 pigs, immediately after withdrawal of the balloon catheter, a3-mm Dispatch catheter (Scimed) with a double lumen was advancedto the site of balloon angioplasty to deliver either saline (vehicle;n54) or FPTIII (25mmol/L; n57) over a 15-minute infusion periodat a rate of 200mL/min (total injection volume 3 mL containing 75nmol FPTIII). The construction of this catheter essentially isolatesthe angioplastied segment of the artery and supplies to the luminalsurface a chosen concentration of drug while permitting blood toflow through the central lumen to supply the distal tissues. Afterremoval of all catheters, the femoral artery was permanently ligatedand the wound to the leg repaired. All animals were given antibiotic(ampicillin 50 mg/kg IM) and analgesic (buprenorphine 0.5 mg IM)cover before recovery from the anesthetic. Four weeks after surgery,the pigs were euthanized with sodium pentobarbitone (70 mg/kg),the heart was removed, and both the entire LAD and left circumflexcoronary artery (LCx) were dissected free. The injured section of theLAD and a segment of LCx of equivalent diameter were each cutinto 4 artery rings. Two of the rings were used immediately forfunctional studies, with the remaining 2 rings placed into phosphate-buffered formal saline for subsequent histological analysis.

Functional AnalysisTwo rings from both the LAD and LCx were mounted on intralu-minal steel wires in Krebs solution aerated with 95% O2/5% CO2 at37°C. The vessels were equilibrated at their optimum resting force of4 g for 1 hour before repeated exposure to 40 mmol/L KCl until 2consecutive applications resulted in identical contractions. One ringfrom each artery was used to construct cumulative concentration-response curves to 5-HT, phenylephrine KCl, calcimycin (A23187),and morpholinosynonimine (SIN-1). Vasorelaxant responses weredetermined after precontraction with 5-HT (1026 mol/L). The re-maining 2 rings were used as time-matched controls to correctrelaxant responses in the case of loss of tone over the course of theexperiment.

Histological Analysis and MorphometryFixed LAD and LCx rings were processed for histological analysisand embedded in paraffin wax. Blocks were coded by an individualwith the research group until histological analysis was complete. The4-mm sections were cut and stained with (1) hematoxylin and eosinfor morphological assessment and (2) alcian blue to detect thepresence of proteoglycans. Planimetry was used to determine therelative areas of the vessel layers from photomicrographs of sectionsfrom each artery. Proteoglycan deposition was quantified by 2independent observers to estimate the percentage of positive stainingwithin the neointima.

MaterialsUnless otherwise stated, chemicals were purchased from SigmaChemical Co. Cell culture materials were purchased from GibcoBRL Life Technologies Ltd. FPTIII was obtained from Calbiochem.Anti–ERK-2 and anti-Ras monoclonal antibodies were purchasedfrom Affiniti. Reporter HRP-linked anti-mouse antibody was a giftfrom the Scottish Antibody Production Unit, Carluke. 1,4-Dithiothreitol and streptavidin-peroxidase were purchased fromBoehringer Mannheim. Amersham International supplied the p42/p44 Biotrak MAPK assays, nitrocellulose membranes, enhancedchemiluminescence detection kit, methyl-[3H]thymidine, and[32P]ATP. Halothane was obtained from Mallinckrodt Veterinary andamphipen LA from Mycofarm UK Ltd.

Statistical AnalysisFor the in vitro biochemical studies, values shown are the mean6SDof n experiments performed on separate cell preparations. For thep42/p44 MAPK activity assay, data were calculated as the activity inpmol · min21 · mg21 cell lysate protein and expressed as the foldincrease above basal MAPK levels. With the [3H]thymidine incor-poration assays, data were expressed as the fold increase above basallevels. Groups were compared by pairedt test or 1-way ANOVAfollowed by Tukey’s test for multiple comparisons. For the in vivostudies, values shown are the mean6SEM of n animals. Morpho-logical data are expressed as area (mm2) and were compared by1-way ANOVA followed by Dunnett’s test. Concentration-responsecurves were compared by 2-way ANOVA followed by a Newman-Keuls post hoc test. In all cases, a value ofP,0.05 was taken toindicate statistical significance.

ResultsEffect of FPTIII on Mitogenic Signal Transductionin Cultured PVSMCsFPTIII concentration-dependently (1 to 25mmol/L) reducedthe p21ras levels in PVSMC membranes (Figure 1), virtuallyabolishing membrane association at 25mmol/L. This isconsistent with an inhibitor action at the level of FTase. In theMAPK activity assay, FPTIII inhibited responses to PDGFand endothelin but not to PMA or bradykinin (Figure 2a).This demonstrates that the effect of FPTIII is restricted togrowth factors (PDGF) and Gi-coupled receptor agonists (eg,ET-1) that signal to p42/p44 MAPK via a p21ras-dependent

Figure 1. Western blot of p21ras in membranes prepared fromPVSMC cell fractions pretreated with FPTIII (1 to 25 mmol/L).Representative of experiments performed on 3 separate cellpreparations.

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pathway. In contrast, p42/p44 MAPK activation in responseto Gq-dependent receptor agonists, such as bradykinin, andprotein kinase C activators, such as PMA, is insensitive toFPTIII, because these agonists regulate p42/p44 MAPK via aRas-independent mechanism in several cell types.14–16 Shiftblots of agonist-induced phosphorylation of p42/p44 MAPKactivity confirmed the differential effect of FPT on responsesto PDGF and PMA (Figure 3).

Thymidine incorporation studies also demonstrated thatincubation of PVSMCs with FPTIII (25mmol/L) for 18 to 24hours inhibited PDGF-induced [3H]thymidine incorporationinto DNA (Figure 4), providing direct evidence that FPTIIIinhibits cell proliferation. Furthermore, the effect of FPTIIIon PDGF-induced MAPK activity was readily reversed by100 nmol/L farnesyl phosphate (FPP substrate for FTase andthus a competitor with FPTIII for FTase; Figure 2b), consis-tent with an action of FPTIII to inhibit FTase.

Effect of FPTIII on Neointima Formation AfterCoronary Arterial Balloon InjuryThe response to injury in control, untreated LADs was similarto that reported in previous studies17 and consisted of the

development of neointimal lesions in close proximity to areasof rupture of the internal elastic lamina (IEL). The lesionscontained large numbers of vascular SMCs, oriented in alongitudinal rather than circumferential pattern, covered withan intact endothelial cell layer (Figure 5b). Intense positiveproteoglycan staining (Figure 5c; 50.066.2% of the neointi-ma) demonstrated production of extracellular matrix. Signif-icant neointimal formation (19.660.8% of total medial andintimal areas) was observed in all untreated porcine LADs.Significant adventitial thickening was also present in thesearteries (Figure 6). Local application of drug vehicle (saline)to the injury site in 4 control pigs did not result in neointimathat was any different in size or morphology from the rest ofthe control group; thus, the planimetric data from all of thecontrol pigs were pooled. In LADs from pigs treated with 25mmol/L FPTIII, there was marked suppression of the extentof neointima formation (Figure 5d) and no adventitial thick-ening (Figure 6), despite rupture to the IEL. Furthermore,significantly less (26.564.6%;P,0.05) positive proteo-glycan staining was present in FPTIII-treated arteries(Figure 5d).

Figure 2. Top, Effects of FPTIII (1 to 25 mmol/L, 18 to 24 hours)on p42/p44 MAPK activation stimulated by PDGF (10 ng/mL;n56), ET-1 (100 nmol/L; n55), PMA (1 mmol/L; n54), and BK(200 nmol/L; n54). Bars: Open, control; right-hatched, 1 mmol/L;solid, 5 mmol/L; left-hatched, 10 mmol/L; and cross-hatched, 25mmol/L FPTIII. Bottom, Reversal of PDGF-induced (10 ng/mL)p42/p44 MAPK activity by farnesyl phosphate (100 nmol/L; n54)added at same time as FPTIII (25 mmol/L). Bars: Open, controlPDGF-stimulated MAPK activity; solid, PDGF- and FPTIII-treatedPVSMCs; and hatched, PDGF- and FPTIII-treated1farnesylphosphate (FPP) replacement. For both panels, *P,0.05 vsagonist-stimulated MAPK activation in absence of FPTIII.

Figure 3. Shift blots of agonist-induced phosphorylation of p42MAPK. Top, Inhibition of PDGF-induced MAPK activation byFPTIII (1 to 25 mmol/L, 18 to 24 hours); bottom, no effect ofFPTIII on PMA-dependent activation. MAPK (lower band) andactivated MAPK (upper band) are denoted by arrows. Represen-tative results of experiment performed on 3 separate cellpreparations.

Figure 4. Effect of FPTIII (25 mmol/L, 18 to 24 hours) on PDGF-stimulated [3H]thymidine incorporation into DNA in PVSMCs(open bar, control; solid bar, FPTIII-treated; *P,0.05 vs PDGFalone).

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Effect of Balloon Injury and FPTIII Treatment(25 mmol/L) on Porcine Coronary ArteryVascular FunctionCumulative concentration-response curves to KCl, 5-HT, andphenylephrine demonstrated an enhanced response to thesecontractile agents in arteries injured 4 weeks previouslycompared with noninjured LCx rings (Figure 7a through 7c).Treatment with FPTIII at the time of injury abolished(P,0.05) the increased responsiveness to 5-HT and phenyl-ephrine and attenuated the enhanced response to KCl. Therelaxant responses of arteries precontracted with 5-HT (1026

mol/L) to both the endothelium-dependent vasodilator (cal-cimycin) and the endothelium-independent vasodilator SIN-1were not significantly impaired by injury (Figure 8), demon-strating that sufficient endothelial regrowth had occurred torestore endothelial function. Importantly, both the endotheli-um-dependent and -independent relaxations in FPTIII-treatedarteries were also intact, demonstrating that the drug had noadverse effect on either endothelial recovery or the ability ofthe coronary artery to dilate.

DiscussionThis study is the first to demonstrate that local short-term (15minutes) application of an FTase inhibitor, targeting thecritical step in adherence of p21ras to the membrane requiredfor MAPK activation,18 prevents neointima formation afterballoon angioplasty while preserving endothelial function.This is consistent with the idea that intervention at the levelof p21ras processing can abolish mitogenic signal transductionin SMCs and with our observations that FPTIII abolishedPDGF-induced p42/p44 MAPK activation, p21ras targeting to

blue demonstrate presence of large pools of proteoglycan (PG;blue) within neointimal lesion of nontreated control artery (C).FPTIII-treated injured artery (D) shows little neointima formed, withonly weak positive PG staining. Bar5100 mm. L indicates lumen;EC, endothelial cell; M, media; and NI, neointima.

Figure 5. Micrographs of transverse sections from noninjured LCx(A) and untreated injured LAD (B), both stained with hematoxylin-eosin. Injured artery exhibits rupture of IEL and marked neointimaformation associated with IEL rupture. Sections stained with alcian

Figure 6. Effect of 15-minute local perfusion with FPTIII (25mmol/L; n57) on neointima size (top) and adventitial thickening(bottom) 4 weeks after balloon injury vs control pigs (n511).*P,0.05 vs noninjured LCx from same animal; #P,0.05 vsinjured artery in control pigs.

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membranes, and thymidine incorporation in cultured porcinecoronary arterial SMCs in vitro. Our findings also extendprevious observations in rat and human primary cell cul-tures8,9 by demonstrating that FPTIII blocks the actions ofagonists other than PDGF (eg, ET-1) that signal throughp21ras but has no effects on agonists that use a mechanismindependent of p21ras.

The primary mechanism through which FPTIII preventsneointima formation is inhibition of early proliferation ofSMCs, according to the data from the thymidine incorpora-tion studies. It is also probable, however, that migration ofSMCs into the neointima may be prevented, as observed withother FTase inhibitors in cultured rat SMCs.8 The FPTIII-induced reduction in proteoglycan is most likely a conse-quence of the smaller neointima size, containing fewer SMCssynthesizing extracellular matrix. FPTIII, however, may alsobe interfering with the signaling pathway involving trans-forming growth factor-b as a mediator in matrix proteoglycansynthesis,19,20 because binding of FTase to type I transform-ing growth factor-b appears to be an essential step in thissignaling process.21 FPTIII inhibited neointimal hypertrophyat 4 weeks after injury, which is the time of maximalneointima development in the balloon-injured pig coronaryartery.17 It would be valuable to have further data with longer

time points to confirm that neointima growth remainssuppressed.

There are several benefits of targeting a common step inthe pathways controlling cell proliferation, such as Rasprocessing. First, it is well recognized that the formation of aneointimal lesion is the result of a complex interplay betweena range of growth stimulants. Therefore, blocking the actionof one mediator is unlikely to prevent proliferation, which canbe triggered by the remaining mediators. Second, FPTIII willtarget only those cells that are actively migrating and prolif-erating while, importantly, having no effect against normallyfunctioning differentiated cells. Our results show that endo-thelial function after FPTIII administration is intact, suggest-ing a “sparing” of endothelial cell regrowth. This may offer amajor advantage over other antiproliferative agents currentlybeing investigated for use in the prevention of restenosis,because there is no evidence in the literature that these agentsalso spare endothelial function. A further advantage of FPTIIIis the preservation of vascular function. FPTIII prevents thehyperresponsiveness to contractile agonists seen after injurywhile preserving normal contractile function. The presence ofa neointimal lesion has been shown previously to result inenhanced sensitivity to 5-HT; thus, the reversal of enhancedcontractility to 5-HT may be merely a result of a reduction inthe amount of neointima present. The present findings with 3different contractile agonists, however, suggest that an addi-tional clinical benefit of a FTase inhibitor may be to reducethe susceptibility of the artery to postangioplasty vesselspasm. In contrast, local paclitaxel administration has beenshown to result in a major impairment of contractile func-tion.22 Finally, the importance of farnesylation of Rho inmediating the migratory responses of SMCs to agonists suchas thrombin is becoming more apparent.23,24Thus, both SMC

Figure 7. Cumulative concentration-response curves to (a) KCl,(b) 5-HT, and (c) phenylephrine in noninjured LCx rings (n;n514), untreated LAD injured 4 weeks previously (‘; n57), andinjured arteries treated with FPTIII at time of injury (’; n57).*P,0.05 vs noninjured LCx rings; #P,0.05 vs untreated injuredLAD rings.

Figure 8. Relaxant response of arteries precontracted with 5-HT(1026 mol/L) to endothelium-dependent vasodilator calcimycin(a) and endothelium-independent vasodilator SIN-1 (b) in nonin-jured LCx rings (n; n514), untreated LAD injured 4 weeks previ-ously (‘; n57), and injured arteries treated with FPTIII at time ofinjury (’; n57). *P,0.05 vs noninjured LCx rings.

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migration and proliferation should be prevented by an FTaseinhibitor. Because FPTIII may act on Rho in addition to Ras,future studies will be needed to explore the relative impor-tance of these 2 actions in neointima development.

Local drug delivery has a number of advantages oversystemic drug delivery for the prevention of restenosis.FPTIII administration reduced adventitial thickening in re-sponse to injury in treated arteries. This has importantimplications, because adventitial hyperplasia is thought toplay a major role in arterial remodeling,25 which also is alimiting factor in the success of clinical angioplasty.26 Be-cause this aspect of the response to injury is often addressedby the use of intracoronary stents, which in themselvesencourage SMC proliferation, the use of local drug deliveryafter injury may preclude the need for this. An additionalbenefit of local delivery is that sufficient concentrations ofdrug can be administered to the vessel wall while the risk ofsystemic side effects is limited.

ConclusionsThe results of the present study show that a 15-minuteapplication of an inhibitor of FTase by local delivery imme-diately after angioplasty has a profound effect on neointimaformation assessed in pigs at a time when the process iscomplete. These findings could have a major impact on thesuccess of clinical vascular reconstructive surgery and de-crease the requirement for second angioplasty intervention orsubsequent coronary bypass, providing a considerable im-provement in the clinical efficacy of current balloon angio-plasty techniques. Another major advantage is that short-termdrug application would preclude the need for prolongedsystemic or local (eg, coated stents) drug administration,which would limit the opportunity for adverse drug reactionsto occur.

AcknowledgmentsDr Work was supported by a Henry Dryerre Royal Society ofEdinburgh Scholarship from the Carnegie Trust for the Universitiesof Scotland. Dr S. Pyne is a Wellcome Trust Senior Research Fellow.We are grateful to Hoechst-Marion-Roussel, Frankfurt, Germany, forfinancial assistance.

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Cherry L. WainwrightLorraine M. Work, Allan R. McPhaden, Nigel J. Pyne, Susan Pyne, Roger M. Wadsworth and

Formation In Vivo After Porcine Coronary Balloon AngioplastyShort-Term Local Delivery of an Inhibitor of Ras Farnesyltransferase Prevents Neointima

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