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Chimeric DNA-RNA Hammerhead Ribozyme to Proliferating Cell Nuclear Antigen Reduces Stent-Induced Stenosis in a Porcine Coronary Model

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Chimeric DNA-RNA Hammerhead Ribozyme to Proliferating Cell Nuclear Antigen Reduces Stent-Induced Stenosis in a Porcine Coronary Model
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  Hidehiko Honda, Raj Makkar, Jack Barber and Frank Litvack Aaron Frimerman, Peter J. Welch, Xiaomei Jin, Neal Eigler, Soonpin Yei, James Forrester, Reduces Stent-Induced Stenosis in a Porcine Coronary ModelChimeric DNA-RNA Hammerhead Ribozyme to Proliferating Cell Nuclear Antigen Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1999 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation doi: 10.1161/01.CIR.99.5.6971999;99:697-703 Circulation. http://circ.ahajournals.org/content/99/5/697 World Wide Web at: The online version of this article, along with updated information and services, is located on the  http://circ.ahajournals.org//subscriptions/ is online at: Circulation Information about subscribing to Subscriptions:  http://www.lww.com/reprints Information about reprints can be found online at: Reprints:  document. Permissions and Rights Question and Answer this process is available in theclick Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located, can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Circulation in Requests for permissions to reproduce figures, tables, or portions of articles srcinally published Permissions:  by guest on February 1, 2014http://circ.ahajournals.org/ Downloaded from by guest on February 1, 2014http://circ.ahajournals.org/ Downloaded from   Chimeric DNA-RNA Hammerhead Ribozyme toProliferating Cell Nuclear Antigen Reduces Stent-InducedStenosis in a Porcine Coronary Model Aaron Frimerman, MD; Peter J. Welch, PhD; Xiaomei Jin, MD, PhD; Neal Eigler, MD;Soonpin Yei, PhD; James Forrester, MD; Hidehiko Honda, MD; Raj Makkar, MD;Jack Barber, PhD; Frank Litvack, MD  Background  —Stent-induced coronary restenosis is a major clinical and public health problem. Proliferating cell nuclearantigen (PCNA) is an important regulator of cell division, and blocking of its expression after angioplasty may limitintimal proliferation.  Methods and Results —We cloned the porcine  PCNA  gene and constructed a chimeric hammerhead ribozyme to asegment of the gene with human homology. In vitro studies with both cultured porcine and human vascular smoothmuscle cells demonstrated uptake of ribozyme within the nucleus and significant inhibition of cellularproliferation. The ribozyme was then delivered locally into pig coronaries in a stent model. At 30 days,histomorphometric analysis showed neointimal thickness of 0.51  0.20 mm in the ribozyme group versus0.71  0.27 and 0.66  0.25 mm in stent controls and scrambled ribozyme control, respectively ( P  0.002,  P  0.03).Quantitative angiographic analysis showed late loss of 1.4  0.5 mm for ribozyme versus 1.9  0.4 and 2.0  0.4 mmfor the controls ( P  0.05 and  P  0.02). Conclusions —Chimeric hammerhead ribozyme to PCNA inhibits smooth muscle cell proliferation in vitro and reducesboth histomorphometric and angiographic restenosis in the porcine coronary stent model when delivered locally. ( Circulation . 1999;99:697-703.)Key Words:  restenosis    stents    oligonucleotides    RNA, catalytic A lthough balloon angioplasty of coronary stenoses re-stores blood flow and relieves symptoms, the rate of restenosis is   35% to 50% in the first 6 months after theprocedure. The principal mechanisms responsible for reste-nosis have been well defined: early elastic recoil, late remod-eling, and intimal hyperplasia. 1–3 The first 2 mechanisms areprevented by coronary stents. On the other hand, stentsincrease the magnitude of intimal hyperplasia. 4 As a conse-quence of the interaction of these opposing factors, the earlyrandomized trials suggest that stents reduce the restenosis rateto the range of 20% to 30%. 5,6 There is, however, noestablished method to prevent the exuberant intimal hyper-plasia that causes in-stent restenosis.Therapeutic interventions that inhibit the activation andproliferation of medial smooth muscle cells should reduceintimal hyperplasia after angioplasty and stent-induced vas-cular injury. 7–9 Cell proliferation can be blocked by a varietyof mechanisms, one of which is inhibition of the expressionof proteins necessary for cell-cycle progression. One suchprotein, proliferating cell nuclear antigen (PCNA), is anattractive target for several reasons. PCNA is a cofactor forDNA polymerase, 10 is required for DNA synthesis andS-phase progression, 11,12 and complexes with other key cell-cycle control proteins, the cyclins and cyclin-dependentkinases. 13,14 In addition, cells undergoing cell-cycle arrest usethe potent cell-cycle inhibitor p21 to bind and inactivatePCNA as a necessary step. 15–18 Finally, there is markedinduction of PCNA expression after balloon injury in the ratcarotid-injury model. 19 Consequently, antisense oligonucleo-tides directed against PCNA have been investigated as atherapeutic agent, not only for prevention of intimal hyper-plasia 20–22 but also for use in other proliferative diseases, 23,24 including cancer. 25 In this study, we examined the hypothesis that ribozymescapable of preventing the initial activation of smooth musclecell proliferation might inhibit intimal hyperplasia. Ri-bozymes are analogous to antisense molecules but possesssome important potential advantages. Like antisense mole-cules, ribozymes specifically base pair with a sequence in thetarget mRNA. Ribozymes, however, have catalytic activity as Received April 1, 1998; revision received September 10, 1998; accepted September 25, 1998.From the Cardiovascular Intervention Center, Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center and the UCLA Schoolof Medicine, Los Angeles, Calif (A.F., N.E., J.F., H.H., R.M., F.L.), and Immusol Inc, La Jolla, Calif (P.J.W., X.J., S.Y., J.B.).Correspondence to Frank Litvack, MD, Cardiovascular Intervention Center, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Suite 6560, Los Angeles,CA 90048.© 1999 American Heart Association, Inc. Circulation  is available at http://www.circulationaha.org 697   by guest on February 1, 2014http://circ.ahajournals.org/ Downloaded from   well, resulting in site-specific cleavage of the target RNAfollowed by release of the ribozyme, which is then free tobind and cleave additional target RNA. Because short-termpersistence of the capacity to cleave target RNA is a centralissue in prevention of intimal hyperplasia, the ability of ribozymes to act substoichiometrically and catalytically is apotential major advantage.In this article, we describe the creation of a chimerichammerhead ribozyme that is directed against PCNA and thattargets a sequence that is conserved in both humans and pigs.We study the effect of this ribozyme on DNA synthesis andsmooth muscle cell growth in vitro. We then use the ribozymein a previously described 26 porcine coronary artery stentmodel to determine its effect on intimal hyperplasia. Methods Design and Creation of a Chimeric HammerheadRibozyme Targeting Porcine and Human PCNA We designed a hammerhead ribozyme capable of recognizing bothpig and human PCNA mRNA. The complete sequence of the humanPCNA cDNA has been previously reported (GenBank accessionnumber J04718). This sequence information was used to clone theporcine PCNA cDNA (Stuart Mahaderiah, PhD, unpublished data,1997). The substrate recognition sequence required by a hammer-head ribozyme is 5  -UH-3  , where H is A, C, or U, with substratecleavage occurring after the H nucleotide (Figure 1). Selection of aneffective ribozyme site also requires that the site be accessible andsingle-stranded in the full-length mRNA. We analyzed several siteswithin human and porcine PCNA mRNA and chose a hammerheadsite (called PCN1) that is 100% conserved between both species. Thesite begins just 12 bp downstream of the AUG protein translationstart site (Figure 1). In vitro cleavage experiments were performedwith full-length PCNA RNA. The PCN1 ribozyme was capable of cleaving both human and porcine targets with equal efficiency.We created a chimeric RNA/DNA hammerhead PCN1 ribozymein which the RNA bases of the substrate binding arms and the stemloop within the ribozyme were replaced with bases of DNA, asshown in Figure 1. Such substitutions would prevent exoribonucle-ase activity and significantly reduce the number of endoribonucleasetargets within the ribozyme. 27,28 Comparison of the chimeric RNA/DNA PCN1 ribozyme with thetraditional “all-RNA” PCN1 ribozyme indicated a significant in-crease in stability within serum (Figure 2). Intact all-RNA ribozymewas undetectable after only 30 seconds in serum (Figure 2, leftpanel), which prevented us from obtaining an accurate estimate of itshalf-life. Intact chimeric RNA/DNA ribozyme, however, was stilldetectable after 5 minutes in serum, with an estimated half-life of 4minutes (right panel). The ribozyme half-life was also similar infreshly prepared human plasma (not shown). Finally, to verify thatthese DNA substitutions did not affect the catalytic activity of theribozyme, in vitro cleavage experiments were performed. No signif-icant difference in activity was observed between the chimeric PCN1and the all-RNA version of the ribozyme. Ribozyme Synthesis and Stability Chimeric RNA/DNA PCN1 hammerhead ribozyme was synthesizedand purified (TriLink BioTechnologies). The functional ribozymecontains the following: (1) 2 8-nucleotide substrate-binding armsconsisting of DNA designed to target PCNA mRNA; (2) thehammerhead catalytic core, consisting of RNA; and (3) the internalhammerhead stem loop, consisting of DNA (see Figure 1). Foruptake studies, a fluorescent tag was covalently attached to the 5  end during synthesis (TriLink BioTechnologies). To evaluate nucle-ase resistance, 15   g of ribozyme (either chimeric or all-RNAPCN1) was incubated in 10% FBS (Gibco BRL) at 37°C for   4hours. Two-microgram aliquots were removed at specific time pointsand transferred immediately to urea gel loading buffer (8 mol/L urea,89 mmol/L Tris, 89 mmol/L boric acid, 10 mmol/L EDTA), heatedfor 5 minutes at 65°C and stored at  70°C. All collected time pointswere then analyzed by denaturing gel electrophoresis and ethidiumbromide staining. Ribozyme degradation and half-life were deter-mined by densitometry (NIH Image software). Cell Culture, Transfection, and Growth Studies Cellular uptake studies were performed with a variety of lipid/ ribozyme combinations to obtain optimal delivery conditions to bothhuman and porcine primary vascular smooth muscle cells in culture.Using fluorescently labeled chimeric PCN1 ribozyme, we estab-lished conditions with the cationic lipid LipofectAMINE (GibcoBRL) that reproducibly yielded delivery to  90% of the cells (datanot shown). To evaluate the efficacy of PCN1 in inhibiting cellularproliferation, ribozyme/lipid complex was first delivered to quies-cent primary cells. After delivery, cells were stimulated with serum,and entry into the cell cycle was monitored by incorporation of [ 3 H]thymidine into newly synthesized S-phase DNA.Low-passage primary human (passages 12 to 14) and porcine(passages 8 to 10) vascular smooth muscle cells were cultured at37°C in DMEM (Gibco BRL) supplemented with 10% FBS, L-gln,sodium pyruvate, and antibiotics. Where appropriate, cells weremade quiescent by culturing in medium containing 0.5% FBS for 48hours. For ribozyme delivery, 1  10 5 quiescent cells were incubatedfor 6 hours with 2.5  g of chimeric ribozyme complexed with 10  gof LipofectAMINE, according to the manufacturer’s suggestions.For cell-growth studies, ribozyme-treated cells were stimulated withfresh media containing 10% FBS and, at the appropriate time points, Figure 1.  PCN1 RNA/DNA chimeric hammerhead ribozyme. The39-nucleotide PCN1 hammerhead ribozyme (uppercase letters)specifically anneals to PCNA cellular mRNA (lowercase letters)and catalyzes the cleavage of the target substrate, as indicated.To protect the ribozyme from ribonucleases, both during deliv-ery and intracellularly, the noncatalytic regions are changed toDNA, as indicated by outlined letters. Figure 2.  Nuclease resistance of RNA/DNA chimeric ribozyme.Fifteen micrograms of PCN1 ribozyme (all RNA, left) or the RNA/ DNA chimeric ribozyme (right) was incubated in 10% FBS for  4 hours at 37°C. Aliquots (2   g) were removed at indicatedtimes and analyzed by denaturing polyacrylamide gel electro-phoresis followed by ethidium bromide staining. Band repre-senting intact ribozyme is indicated. 698  PCNA Ribozyme Reduces Stent-Induced Stenosis  by guest on February 1, 2014http://circ.ahajournals.org/ Downloaded from   pulse labeled with [ 3 H]thymidine (Amersham). After 1-hour of labeling, cells were rinsed with PBS and lysed in 500   L of 0.1mol/L NaOH, 10 mmol/L EDTA, and 0.5% SDS. Genomic DNAwas precipitated at 4°C by addition of 500   L of 20% TCA. Pelletswere washed once with cold 10% TCA followed by cold 70%ethanol. Dried pellets were resuspended in 100   L of 0.1 mol/LNaOH and incorporated [ 3 H]thymidine was measured by scintillationcounting. For detection of fluorescent ribozyme uptake, rat aorticsmooth muscle cells grown on tissue culture chambers were serumstarved for 48 hours, followed by incubation with a mixture of 4   mol/L of fluorescent-labeled PCNA ribozyme and 2   mol/L of lipofectin (Gibco BRL catalog No. 18292-037). After 30 minutes’incubation, FBS was added to 5%, and the cells were continuouslyincubated at 37°C for 48 hours. After being washed in PBS for 3minutes, the cells were observed and photographed with a fluores-cent microscope (Olympus Laborlux S). Porcine Model of Injury-InducedIntimal Hyperplasia To evaluate the effectiveness of PCN1 for inhibiting vascularintimal hyperplasia, stents were placed in balloon-injured coro-nary arteries of normolipemic adult farm pigs. Before stentplacement, the pigs were treated with either (1) PCNA ribozymeinfusion, (2) scrambled ribozyme infusion as control, or (3) noinfusion as control (stent only). Normolipemic adult farm pigsweighing 25 to 30 kg were used. The 18 pigs were fasted the daybefore the procedure and pretreated with oral aspirin (325 mg)and diltiazem (120 mg). The 3 pig groups were as follows: PCNAribozyme (9 arteries: 4 left anterior descending coronary arteries[LAD] and 5 right coronary arteries [RCA]), scrambled ri-bozymes (8 arteries: 4 LAD and 4 RCA) and stent alone (9arteries: 3 LAD, 2 left circumflex [LCx], and 4 RCA). The pigswere anesthetized with intravenous xylazine and ketamine, thenintubated. Anesthesia was maintained with isoflurane. Bretyliumtosylate (5 mg/kg IV) and aspirin (10 mg/kg IV) were adminis-tered. An 8F sheath was inserted in the left carotid artery bycutdown, and 10 000 U of heparin was injected. The left and rightcoronary arteries were cannulated with an 8F AL1.75 guidingcatheter, 200 mg of nitroglycerin was injected, and baselineangiography was performed. A segment of the LAD, LCx, orRCA ranging from 2.7 to 3.5 mm in diameter was selected as atreatment site with the aid of on-line quantitative digitalangiography.After angiography was performed, 1 to 2 arteries per pig wereselected for ribozyme infusion and stent placement. First, arterialwall injury was performed by balloon inflation in the selectedartery segment with a 1.1:1 to 1.2:1 balloon-catheter–to–artery-diameter ratio at 8 atm for 30 seconds. Next, the ribozyme (orcontrol) was injected into the artery wall with a multilumen sleevethat tracks over a standard dilating balloon catheter (LocalMedInfusasleeve II). The Infusasleeve has 4 delivery channels, eachof which has 9 holes for the drug to exit. The sleeve is alignedover the balloon, and the balloon is inflated, injecting the drugunder pressure into the vessel wall. The 5-mL solution, containing180   g of either PCNA or scrambled ribozyme, was then injectedinto the artery wall at 80 to 110 psi for   20 seconds. After theribozyme injection, the balloon and infusion sleeve were pulledback, and intracoronary nitroglycerin was injected. A 15-mm-long balloon-mounted nitinol stent was passed over a 0.014-mmguidewire, with the location of reference side branches used as aguide to position the stent exactly at the ribozyme-infusion site.The balloon was inflated twice to 8 to 11 atm for 20 seconds todeploy the stent. The balloon was pulled back, and a finalangiogram was obtained. Using the computerized quantitativecoronary analysis (QCA) system (Advantx DX, GE MedicalSystem), we measured the treated segment before stent deploy-ment, stenting balloon size, and final in-stent mean diameter. Atany stage of the procedure when coronary spasm was observed,additional intracoronary nitroglycerin (100 to 200   g) wasinjected. The carotid incision was repaired, and the animalrecovered from anesthesia. No other anticoagulant therapy wasgiven. Oral aspirin, ticlopidine, and diltiazem were maintained forthe study duration. Quantitative Coronary Angiography Coronary angiograms were obtained for computerized quantitativecoronary measurements (Advantx DX, GE Medical System) of thetreated segment before and immediately after stent deployment and4 weeks later, before euthanasia. From several orthogonal views, theend-diastolic frame with the worst arterial narrowing was chosen forQCA analysis. The following measures were obtained: mean diam-eter of the proximal and distal segments adjacent to the stented site,in-stent minimal lumen diameter (MLD), and percent diameterstenosis (1 minus MLD divided by reference diameter). Late lumenloss was calculated as the difference between in-stent diameter atplacement and late MLD. Animal experiments conformed to guidingprinciples of the American Physiological Society and were approvedby the Cedars-Sinai Medical Center Institutional Animal Care andUse Committee. Figure 3.  Inhibition of cellular DNA synthesis by PCN1. Quiescent primary human (A) or porcine (B) vascular smooth muscle cells weretreated with liposomes containing chimeric PCN1 hammerhead (PCN1-HH) ribozyme or a nonspecific oligonucleotide (oligo) control,followed by stimulation with 10% serum. DNA synthesis was measured by [ 3 H]thymidine incorporation at 24, 42, and 48 hours afterstimulation. Experiments at each time point were performed on duplicate days. As a comparison for complete lack of cell proliferation,1 subset of cells was not stimulated with serum. Each time point represents experiments done in triplicate. Frimerman et al February 9, 1999  699  by guest on February 1, 2014http://circ.ahajournals.org/ Downloaded from   Histomorphometric Analysis After the final angiogram was obtained, the stented segments of thecoronary artery were removed en bloc. Special histological process-ing was performed to maintain the vascular architecture with metallicstruts in situ. Tissue blocks were cut with a diamond wafering bladeand embedded in methyl methacrylate. Four radial cross sectionscontaining 18 struts 3 mm apart were cut from each stent-containingsegment. Sections were ground to a thickness of 30 mm, opticallypolished, and stained with toluidine blue (paragon stain). Sectionswere analyzed with a computer-assisted morphometric program(Optimas Inc). The cross-sectional areas of the lumen, neointima,vessel within the boundaries of the stent, and external elastic lamina(EEL) were measured. Regional neointimal thickness (NIT) wasmeasured for every stent strut. The corresponding depth of strutinjury was scored as described by Schwartz et al, 4 where 0  internalelastic lamina (IEL) intact, 1  IEL fractured by strut, 2  strut Figure 4.  Fluorescent micrograph of porcine vascular smooth muscle cell 48 hours after incubation with chimeric fluorescent ribozymeto PCNA. Fluorescence within the cell nucleus is seen. Figure 5.  Top, Low-power (   10) cross sections of porcine arteries at 4 weeks after stent implantation. A (stent only) and B (scrambledribozyme) demonstrate exuberant intimal hyperplasia within the struts. C (ribozyme) shows a substantially lesser magnitude of hyper-plasia. Bottom, Corresponding high-power magnifications. No histological evidence of tissue inflammation is seen. 700  PCNA Ribozyme Reduces Stent-Induced Stenosis  by guest on February 1, 2014http://circ.ahajournals.org/ Downloaded from 
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