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J Thorac Cardiovasc Surg 1998;115:152-161
© 1998 Mosby, Inc.

SURGERY FOR CONGENITAL HEART DISEASE

Saphenous vein graft protection: Effects of c-myc antisense

From the Cardiovascular Research Center, Departments of Medicine (Cardiology)^a and Surgery (Cardiothoracic Surgery),^b Thomas JeffersonUniversity, Philadelphia, Pa., and Clinica Olivos, Buenos Aires, Argentina.^c Supported by Lynx Therapeutics, Hayward, Calif

Received for publication June 24, 1997. Accepted for publication August 29, 1997. Revisions requested August 12, 1997; revisions received August 27,1997. Address for reprints: Andrew Zalewski, MD, Thomas Jefferson University,Cardiovascular Research Center, Division of Cardiology, Suite 410N, 1025 WalnutSt., Philadelphia, PA 19107.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective:Saphenousvein grafting is associated with extensive medial remodeling, characterizedby cellular proliferation, loss of smooth muscle cells, and an inflammatoryresponse. In this study, we examined whether unfavorable responses to veingrafting could be modified by the intraoperative application of c-myc antisense oligomers.
Methods: The intragraft cell proliferation,macrophage infiltration, and medial preservation were examined in a porcinemodel in the control and antisense-treated groups (n = 36).
Results: Saphenous veins showed transmuraldistribution of oligomers within 30 minutes of the ex vivo incubation. A concentration-dependentinhibition of cell proliferation in the media of saphenous grafts was noted3 days later (0 to 200 µmol/L,p = 0.005). The growth inhibition was sequence-specific, becausecontrol oligomers produced only insignificant effects (20 µmol/L). Vasculareffects of c-myc antisense were associated with a significant attenuation of macrophage infiltrationin saphenous grafts. A concentration-dependent decrease in tissue edema (p = 0.0005)and the attenuated loss of smooth muscle cells (p = 0.002) were noted in the media ofthe arterialized venous conduits after c-myc antisense.
Conclusions: Direct application of syntheticDNA to harvested saphenous veins resulted in a rapid transmural distribution.The inhibition of the intragraft cell proliferation in vivo after c-myc antisense was sequence dependent.Decrease in vein graft injury resulted in an attenuated inflammatory responseand better medial preservation. These findings provide a rationale for assessmentof the long-term effects of vein graft protection with c-myc antisense.

	Introduction

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Bypass grafting with autologous saphenous vein has become the cornerstoneof therapy for obstructive coronary artery disease since its introductionby Favaloro in 1969. ¹ Clinicaloutcomes of this procedure, however, are marred by a progressive loss of saphenousvein graft (SVG) patency, which is particularly accelerated at later years. ^2,3 The preexisting changes in saphenous vein may have an impact on thefate of SVGs. ⁴ More important,however, vascular injury is commonly induced by grafting. ^5-7 These changesare accompanied by the rapid activation of immediate-early gene expression,including c-fos and c-myc protooncogenes, and contribute to cell proliferation. ⁸ Subsequent events, including thrombosis,intimal formation, and even late graft atherosclerosis, can be viewed as theresponse to ubiquitous vascular injury that develops after vein arterialization.

Our previous observations indicated the presence of intragraft cellproliferation and suggested migration of myofibroblasts through the injuredmedia to neointima. ⁹ The accompanyingchanges resemble the process of wound healing and may result in unfavorableremodeling of the conduit. ^10,11 The intima, which often replaces damaged media,becomes the nidus for future atherosclerotic plaque. ¹² On the basis of these observations, we examinedthe hypothesis that early changes in SVGs can be attenuated by the intraoperativeapplication of an intervention aimed at controlling cell proliferation. Inthis study, we report concentration-dependent effects of synthetic antisenseDNA directed against the c-myc protooncogenein porcine SVGs. The results demonstrate reduction in cell proliferation andimproved preservation of the media in SVGs. These data provide a rationalefor the future examination of this strategy to improve the long-term patencyof SVGs.

	Methods

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Animal preparation.
A porcine model of SVG interposition in the common carotid artery wasused. ^7,9,10 Aftermedication with aspirin (650 mg orally) and cefazolin (1 gm intravenously),crossbred pigs (n = 36, 22 to 55 kg)were sedated with ketamine (20 mg/kg intramuscularly) and xylazine (4 mg/kgintramuscularly). The anesthesia was maintained with endotracheal halothane(0.75%) and oxygen. Lateral saphenous veins (6 to 10 cm) were isolated andthen incubated in a saline solution bath (total volume 10 ml) with eitherno oligomers or various sequences, as described later. The carotid arterieswere dissected free and heparin sodium (150 units/kg intravenously) was administered.After a 2 cm portion of the carotid artery was excised, the proximal end wasanastomosed with a continuous suture, and a similar procedure was performedon the distal anastomosis. The incision was closed, and the animals were allowedto recover. Replicating cells in SVGs were labeled in vivo with an intravenousinjection of 5-bromo-2'-deoxyuridine (30 mg/kg BrdU; Boehringer MannheimCorp., Indianapolis, IN) 2 hours before the animals were euthanized.

The animals were euthanized with an intravenous overdose of Euthasol(Delmarva Laboratory, Midlothian, VA) containing pentobarbital sodium (1950mg) and phenytoin sodium (250 mg) 3 days after the operation. All procedureswere in accordance with institutional guidelines and in compliance with the "Guidefor the Care and Use of Laboratory Animals" prepared by the Instituteof Laboratory Animal Resources and published by the National Institutes ofHealth (NIH Publication No. 86-23, revised 1985).

Oligomer preparation and application.
Phosphorothioate oligomers were synthesized by an automated deoxyribonucleicacid (DNA) synthesizer and purified by high-pressure liquid chromatography(Lynx Therapeutics, Hayward, CA). Table 1 depicts oligomer sequencesthat were used in this study. For vascular distribution studies, antisense oligomerswere labeled with carboxyfluoresceins as described by the manufacturer (AppliedBiosystems, Foster City, CA). Freshly excised saphenous veins were then incubatedwith oligomers for 2 seconds, 30 minutes, and 1 hour. For in vivo studies,individual saphenous veins were randomly assigned to different concentrationsof antisense DNA (0, 2, 20, and 200 µmol/L)or different oligomer sequence (no oligomer, antisense, scrambled, or mismatched)as indicated in the text. After the 30-minute incubation period in the respectivesolution, saphenous veins were interposed in the carotid artery and the remainingbath solution was topically applied to the perigraft region.

In the remaining experiments, carotid arteries were exposed at the timeof SVG harvesting. Distal ends were opened to demonstrate pulsatile bloodflow and ensure graft patency in situ. Occluded vessels were excluded fromfurther analysis (0, 3, 2, and 4 in 0, 2, 20, and 200 µmol groups, respectively, not significant), whereas patentvessels were excised with surrounding tissues and immersed in HistoChoicefixative (Amresco, Solon, OH) for at least 5 hours. They were sectioned (5mm blocks), processed in a TissueTek vacuum infiltration processor (MilesScientific, Naperville, IL), and embedded in paraffin. The sections (5 µmthick) from each block were stained with Verhoeff's stain to determine theoverall vessel structure. Multiple sections from at least three blocks representingthe body of the graft were selected for this study. The n values reported in the Results section represent the number ofvessels.

Immunohistochemistry.
The Vectastain Elite ABC system (Vector Laboratories, Inc., Burlingame,CA) was used as previously described. ⁹ The following primary antibodies were used: a monoclonal mouse antibodyrecognizing BrdU (1:200, Novocastra Laboratory, Ltd., Newcastle Upon Tyne,UK), a monoclonal mouse DE-R-11 antibody recognizing intermediate filamentdesmin (1:50; Novocastra); a monoclonal mouse antiporcine macrophage immunoglobulinG_2b antibody (1:10, ATCC HB 142.1; American Type Culture Collection,Rockville, MD). Afterward, slides were incubated with biotinylated secondaryhorse antimouse immunoglobulin G (1:2000; Vector Laboratories) for 1 hour.They were then stained with diaminobenzidine tetrahydrochloride substratekit (Vector Laboratories) followed by counterstain with Gill's hematoxylin(Sigma Diagnostics, St. Louis, MO). Positive control preparations for desminincluded sections of porcine coronary arteries and normal human saphenousveins. For the antimacrophage antibody, positive control preparations consistedof lung alveolar macrophages. Negative control procedures were carried outwith nonimmune serum instead of primary antibody.

Data analysis.
To examine concentration- or sequence-dependent effects of oligomerson cell proliferation, cells showing BrdU in at least three different sectionsfrom each graft were counted without the knowledge of random assignment. Toavoid selection bias, quantitative measurements were carried out in the entiremedia under the same magnification; mean values were then calculated. Histologicmarkers of vein graft injury (vessel morphometry, desmin immunostaining, macrophagecount) were analyzed in serial sections adjacent to those with the highestnumber of BrdU-labeled cells in each vessel to account for the focal changesafter vein grafting. To quantify medial changes (vessel morphometry, desminimmunostaining), the sections were digitized with a Sony DXC-750MD video camera(Sony Corporation, Tokyo, Japan) attached to a Nikon Optiphot-2 microscope.To obtain medial areas, sections stained with Verhoeff's stain were then processedwith a video analysis software package (Media Cybernetics, Silver Spring,MD). To quantify the loss of medial smooth muscle (SM) cells after grafting,digitized images of desmin immunostaining were analyzed with the Image-Prosoftware (Media Cybernetics). From each vessel, a microscopic field exhibitingthe worst medial damage was selected. The area occupied by desmin was thenautomatically quantified and expressed as the percentage of media.

Numeric data are presented as mean ± standard error. Todetermine whether the effects of c-myc antisenseDNA were concentration dependent, statistical significance was calculatedwith linear regression analysis. Furthermore, analysis of variance was carriedout to examine the overall differences in multigroup comparisons. If a significant p value was present, Bonferroni correction was usedto identify specific intergroup differences. A pvalue of less than 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Distribution of antisense DNA in saphenous vein conduits.
To examine the distribution and time course of oligomer uptake in saphenousveins, freshly harvested segments were incubated ex vivo with fluorescent-labeledantisense DNA (200 µmol/L) for 2 seconds,30 minutes, and 1 hour. As shown in Fig. 1, transmural distribution of oligomerswas apparent after 30 minutes. Nuclear localization of the label pointedto the intracellular uptake of antisense DNA. A similar transmural distributionof oligomers was also evident by direct perivascular application of oligomersto the vessels in situ (not shown), which suggested that the pattern of oligomeruptake was not related to tissue damage associated with vein isolation. Onthe basis of these results, the 30-minute incubation period was selected forthe remaining in vivo experiments.

View larger version (77K): [in this window] [in a new window]   Figure 1. Time-dependent vascular distributionof fluorescent oligomers. Human saphenous vein segments were incubated exvivo with c-myc antisense DNA (200 µmol/L) and examined by fluorescent microscopy. A, At 2 seconds, fluorescence is visible only along luminaland adventitial surfaces, whereas the media (m)is devoid of oligomers. Note the absence of autofluorescence in the media. B, At 30 minutes, transmural distribution of oligomersis associated with nuclear localization of fluorescence. C, At 1 hour, the media (m) isfurther saturated with labeled oligomers. Experiments were repeated threetimes, yielding similar results. (Magnification x25.)

View larger version (136K): [in this window] [in a new window]   Figure 2. Cell proliferation in porcineSVGs. Harvested saphenous veins were treated with no oligomers (A) or with c-myc antisense DNAat concentrations of 2 µmol/L (B), 20 µmol/L (C), or 200 µmol/L (D). Proliferating cells were labeled in vivo with BrdUat 3 days after vein interposition into the carotid arteries. BrdU-labeledcells (brown nuclei) are decreased in the media (m)of SVGs treated with 20 µmol/L and 200 µmol/L of c-mycantisense DNA. Arrows point to the border between the media (m) and adventitia. Labeled cells outside the media represent proliferatingcells in the adventitia and perivascular wound. (Magnification x150.)

View larger version (41K): [in this window] [in a new window]   Figure 3. Bar graph illustrating reductionin medial cell proliferation after c-myc antisenseDNA in SVGs. Results are presented as mean ± standard error, with p values referring to comparisons between treatmentand control (no oligomers) groups.

View larger version (79K): [in this window] [in a new window]   Figure 4. Inflammatory respose in SVGs. A, Untreated saphenous veins exhibited focal accumulationof macrophages in the media (dark brown staining). B, Treatment with c-mycantisense oligomers (200 µmol/L) wasassociated with attenuated inflammatory response 3 days after the operation.Note the decrease in infiltrating macrophages in the media of the graft, whereasadhering macrophages at the luminal side were less affected. Arrows point to the border between the media and adventitia. Labeledcells outside the media represent macrophages in the adventitia and perivascularwound. (Magnification x 150.)

View larger version (27K): [in this window] [in a new window]   Figure 5. Bar graph illustrating changesin inflammatory response in SVGs after treatment with c-myc antisense oligomers. Total macrophage content and infiltratingmacrophages in the media were significantly reduced in treated groups. Thenumber of adhering macrophages, although reduced, did not reach statisticalsignificance. Results are presented as mean ± standard error,with p values referring to comparisons between control (oligomers) and individualtreatment groups (20 µmol/L and 200 µmol/L).

View larger version (80K): [in this window] [in a new window]   Figure 6. Photomicrographs showing themedia of SVGs (Verhoeff's stain). A, Untreatedgrafts demonstrated medial edema, focal necrosis, and, frequently, adheringlayer of thrombus. Note disorganized cells in the media. B, Treatment with c-myc antisenseoligomers (200 µmol/L) was associatedwith reduction of medial edema and overall better preservation of SM cellsin the graft. Note circumferential alignment of SM cells in the media. Arrows point to the border between the media (m) and adventitia. (Manification x 150)

View larger version (62K): [in this window] [in a new window]   Figure 7. Bar graph depicting changesin medial areas of SVGs after c-myc antisenseoligomers. Medial areas are significantly smaller in the treated group (200 µmol/L) as a result of marked reduction in tissueedema and inhibition of inflammatory response. Results are presented as mean ±standard error, with p values referring tocomparisons between treatment and control (no oligomers) groups.

View larger version (83K): [in this window] [in a new window]   Figure 8. SM cell preservation in SVG. A, Untreated grafts demonstrated focal loss of SM cellmarker, desmin. Remaining medial SM cells (brown cytoplasmic stain) are interspersedwith cell debris and nonmuscle cells. B, Markedretention of SM cells in the media was present in SVGs treated with c-myc antisense oligomers (200 µmol/L). Note the paucity of tissue edema and restored microarchitectureof the vein. Arrows point to the border between the media (m) and adventitia. (Magnification x150.)

View larger version (38K): [in this window] [in a new window]   Figure 9.Bar graph showing retentionof SM cells in SVGs after treatment with c-mycantisense oligomers. In areas with maximal histologic medial injury, desmindistribution was quantified. In the antisense-treated group (200 µmol/L), SM retention was significantly improvedcompared with untreated grafts. Results are presented as mean ±standard error, with p values referring tocomparisons between control (no oligomers) and individual treatment groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

Sequence-dependent effects of "antisense" DNA.
Vascular applications of antisense have been based on a premise thatsynthetic DNA complementary to messenger ribonucleic acid (mRNA) of a cell-cycle–regulatinggene can block the proliferative reaction by means of DNA-mRNA hybridization. ^13,14 It should be emphasized, however, that antisense DNA oligomersare pleiotropic compounds that may exert their biologic effects through severalmechanisms. Phosphorothioate backbone prolongs oligomer bioavailability, butit may also contribute to effects unrelated to oligomer-mRNA hybridization. ¹⁵ It is important to recognize thateven some sequence-dependent phenomena are related to interactions with growthfactors (e.g., basic fibroblast growth factor), thereby augmenting antiproliferativeeffects. ¹⁶ This study demonstratedsequence-dependent reduction in medial cell proliferation in SVGs, whereasscrambled phosphorothioate sequence exhibited no effects in vivo (Table II). These findings underscore the dichotomy betweenin vitro and in vivo systems, with the former more likely to elicit sequenceindependent effects. ^15-17 We also tested a mismatched sequence containing4G motif, notorious for several nonantisense effects in cell culture. ^17,18 When different oligomers were compared in vivo with approximatelythe median effective concentration, control sequence with 4G motif did notdemonstrate a significant decrease in cell proliferation. Accordingly, theantiproliferative effects of antisense DNA in vivo cannot be attributed solelyto the presence of contiguous guanosine residues. However, the present findingsdo not exclude the possibility that 4G motif confers additive antiproliferativeeffects at higher concentrations.

The intracellular accumulation of intact anti­sense oligomersis associated with downregulation of c-mycexpression in cells derived from saphenous veins. ¹⁴ Nonetheless, because the c-myc gene represents a common final pathway in the growth regulationof vascular cells, a wide range of other growth inhibitors may reduce itsexpression without direct interactions with target mRNA. ¹⁹ Accordingly, downregulation of the target mRNA orprotein is not sufficient to discern between overlapping antisense (oligomer-mRNAhybridization) and nonantisense (e.g., growth-factor inhibition) mechanisms.Taking into consideration these observations, we prefer to describe the effectsof c-myc antisense oligomers as sequence dependent,rather than categorically describe them as purely antisense.

The role of media in vascular response to injury.
The observed changes in the SVGs have underscored a rapid developmentof medial injury, consistent with previous experimental and clinical findings. ^6,20 The activation of vascular fibroblasts with their differentiation tomyofibroblasts develops after severe medial injury in coronary arteries. ^21,22 Although the changes in SVGs differ from the arterial responseto injury, some aspects of vascular repair are common. ²³ Both perivascular and medial fibroblasts contributeto the repair of porcine arterialized saphenous veins translocating to theneointima. ⁹ Likewise, humansaphenous veins in organ culture preferentially acquire neointima from theadventitia or the edges of broken media (repositories of vascular fibroblasts),but not from the intact media, despite stimulation with growth factors. ²⁴ These observations underscorethe notion that the preservation of the media in SVGs may have an impact onlong-term patency.

The origin of medial damage after saphenous vein grafting is multifactorial.In addition to tissue injury during surgical harvesting, the activation ofcells in situ and medial infiltration with blood-borne inflammatory cellsmay induce local cytotoxic effects. The latter is particularly important becauseinvading macrophages often perpetuate tissue damage. ²⁵ A highly distinct mechanism leading to medial lossmay be represented by apoptosis, which colocalizes in the regions of highproliferation and c-myc expression. ²⁶ The results of this study demonstratethat c-myc antisense DNA attenuates severalmarkers of tissue damage after SVG arterialization. The improvement in histologicpreservation of medial SM cells, paralleled as it is by the decrease in macrophageinfiltration, points to the possibility of vascular protection after arterialization.

Antisense DNA as a therapeutic agent.
Antisense DNA sequences against several growth-regulatory genes havebeen envisioned for the prevention of coronary restenosis. ^27,28 We previouslyshowed the reduction in neointimal formation after antisense oligomers againstthe c-myc in a porcine model of coronary injury. ²⁸ This approach, however, is hinderedby a low efficiency of transcatheter delivery (<1%). In contrast, directex vivo application of antisense DNA to the SVG results in the uniform distributionof oligomers after 30 minutes of incubation, involving not only the adventitiabut also the entire media. This is particularly important for SVG, becausethe events in the media may contribute to late graft failure. Because thetreatment of saphenous veins occurs ex vivo, the risk of potential side-effectsin regions remote from the site of application of antisense DNA is probablyminimized. Furthermore, it is noteworthy that identical antisense DNA hasdemonstrated no significant adverse effects even after the intracoronary administrationin patients after coronary angioplasty. ²⁹ The question of whether the short-term effects of c-myc antisense, as demonstrated in the current study, will translateto a long-term benefit regarding remodeling of the arterialized grafts requiresseparate studies. It should be underscored, however, that medial injury appearsto occur early, so SVG protection may not necessarily require prolonged orrepeated drug administration. Alternative approaches may include attemptsto prolong the bioavailability and cellular uptake of oligomers by complexingthem with liposomes. ³⁰

In conclusion, antisense DNA against the c-myc protooncogene can easily be delivered to harvested saphenous veinsused for arterial revascularization, thus achieving the uniform distributionof oligomers in the target vessel. This results in significant inhibitionof cell proliferation and the reduction in medial damage that develops earlyin SVGs soon after grafting. The antiproliferative effects of antisense DNAare concentration and sequence dependent. These findings provide the basisfor further studies of therapeutic potential of this approach to prevent long-termfailure of SVGs.

We are grateful to Tim Geiser, PhD, and Jerry Zon, PhD, for their insightfulcomments during this study and expert advice concerning oligomer design. Weacknowledge excellent technical assistance of Dian Wang, BS, regarding immunohistochemistry.

	References

Top Abstract Introduction Methods Results Discussion References

 

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