Skip Navigation

Cardiovascular Research 1999 44(1):81-90; doi:10.1016/S0008-6363(99)00182-0
© 1999 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Hughes, G.C.
Right arrow Articles by Landolfo, K. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hughes, G.C.
Right arrow Articles by Landolfo, K. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1999, European Society of Cardiology

Transmyocardial laser revascularization limits in vivo adenoviral-mediated gene transfer in porcine myocardium{star}

G.Chad Hughesa,*, Brian H. Annexb, Bangliang Yina, Anne M. Pippenb, Pengnian Linb, Alan P. Kypsona, Kevin G. Petersb, James E. Lowea and Kevin P. Landolfoa

aDivision of Cardiovascular and Thoracic Surgery, Box 3857, Duke University Medical Center, Durham, NC 27710, USA
bDivision of Cardiology, Box 3857, Duke University Medical Center, Durham, NC 27710, USA

* Corresponding author. Tel.: +1-919-684-5059; fax: +1-919-681-7054 chadh{at}acpub.duke.edu

Received 26 November 1998; accepted 14 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Transmyocardial laser revascularization (TMR) is emerging as a potential treatment option for patients with end-stage CAD, and adjuvant gene therapy may be helpful in further improving the results of the procedure. However, the effects of TMR on gene transfer are unknown. Methods: Swine underwent left thoracotomy. TMR was performed to create five channels at 2-cm intervals in the anterolateral free wall of the left ventricle (LV) followed by injection of 1x109 plaque-forming units (pfu) of a replication-deficient adenovirus vector carrying the reporter gene β-galactosidase (Ad.Pac β-gal). An additional five direct injections of 1x109 pfu Ad.Pac β-gal were made at 2-cm intervals in the posterolateral LV of each heart. Control animals underwent TMR alone/vehicle alone (n=3) or empty virus alone/no treatment (n=3) of the anterolateral/posterolateral LV, respectively. Results: ELISA revealed significantly greater transgene expression in the direct Ad.Pac β-gal injection versus TMR plus Ad.Pac β-gal inject regions at both 3 (n=6) (273.0±58.5 vs. 133.4+28.1 pg β-gal/g protein, P=0.02) and 7 days (n=6) (180.0+59.9 vs. 56.7+18.1 pg β-gal/g protein, P=0.02) postoperatively. At 14 days postoperatively (n=2), no transgene expression was detected in either region. No transgene expression was detected in any of the control regions at 3 days postoperatively. CD-18 staining revealed significantly greater inflammation in the TMR plus Ad.Pac β-gal and TMR alone regions as compared to Ad.Pac β-gal or vehicle (P<0.001). Conclusions: Adenoviral-mediated gene transfer in conjunction with TMR is possible, although TMR appears to limit the degree of transgene expression attained as compared to direct intramyocardial injection alone, likely due to the greater immune response observed with the former. These findings may have important implications for therapeutic strategies aimed at combining TMR with gene therapy for CAD.

KEYWORDS Cardiovascular surgery; Coronary disease; Gene therapy; Gene expression; Inflammation; Swine


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Both transmyocardial laser revascularization (TMR) and gene therapy are emerging as potential treatment options for patients with coronary artery disease not amenable to standard revascularization procedures such as coronary artery bypass grafting or percutaneous angioplasty. TMR uses laser energy to create transmural channels from the epicardial to endocardial surface of the left ventricular free wall in regions of chronically ischemic yet viable myocardium. Clinical studies of TMR in patients with end-stage coronary disease have demonstrated improvements in anginal scores associated with increased perfusion [1–4] and function [5] 3 to 12 months postoperatively. Angiogenesis is one of the presumed mechanisms for the observed clinical response to TMR, and work from our laboratory has demonstrated direct evidence for neovascularization 6 months following the procedure in a model of hibernating myocardium [6].

However, not all clinical studies have demonstrated improved perfusion postoperatively [7–9], and experimental work has shown that although perfusion is improved 6 months following the procedure, levels of resting myocardial blood flow do not return to normal [10]. In addition, clinical improvement following the procedure is typically delayed by several months [1–4], possibly due to the time needed for neovascularization to develop [6]. Consequently, there exists potential for combination therapy with TMR and angiogenic growth factors to further improve on the results of the procedure. Direct myocardial gene transfer involves the introduction of new genetic information into cardiomyocytes to achieve a desired therapeutic effect. Gene transfer of angiogenic growth factors has improved perfusion and function in animal models of myocardial ischemia [11,12]. Although gene therapy has yet to be used to effect improvement in the ischemic human heart, the use of angiogenic proteins in humans has begun [13] with gene transfer techniques on the horizon.

Methods available for in vivo gene transfer include direct injection of naked DNA into tissue [14], injection of DNA complexed with liposomes [15], and infection with recombinant viruses [16]. However, in the cardiovascular system, the most efficient and effective method of in vivo gene transfer to date has been achieved using recombinant adenoviruses [17]. Replication-deficient adenoviral vectors have been used to transfer the complementary DNA (cDNA) for angiogenic polypeptides into ischemic myocardium and appear safe for clinical use [16]. Methods available for delivering vector include intravenous, intracoronary, and direct intramyocardial injection among others [18]. Direct intramyocardial injection of vector may be easily performed at the time of surgery, yet little is known about the potential for combining TMR with gene transfer for the treatment of coronary artery disease. Consequently, the purpose of this study was to investigate the feasibility of performing adenoviral-mediated gene transfer via direct intramyocardial injection in conjunction with TMR, as well as the efficiency of gene transfer and expression in TMR treated myocardium.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Adenoviral vectors
Adenoviral vectors (Ad.Pac β-gal) derived from the in340 mutant strain of adenovirus type 5 were generated as previously described [19,20]. Ad.Pac β-gal contains a nuclear localizing β-galactosidase cDNA in the E1 cloning site with a CMV early enhancer-promoter and an SV40 VP 2 polyadenylation signal. Control empty viral vectors (Ad.null) were constructed in an identical manner except that no transgene was placed in the E1 position. High-titer purified stock of viral vector was generated by infecting 150-mm plates of confluent 293 cells in Dulbecco’s modified Eagle medium (DMEM) containing 2% fetal bovine serum at a multiplicity of infection of 1. After observation of a cytopathic effect, cells were harvested and the virus purified on a cesium chloride gradient using modifications of existing methods [20,21]. Briefly, infected 293 cells were washed from the plates, pelleted by centrifugation at 500 g, and lysed by sonication in virus storage buffer (VSB; 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2). Cell debris was pelleted, and the supernatant adjusted to a density of 1.1 g/ml by addition of solid cesium chloride. This solution was layered onto 1.3–1.4 g/ml cesium chloride step gradient and ultracentrifuged at 100 000 g for 2.5 h at 4°C. The visible band of pure virus was harvested and desalted by serial gel filtration on Sepharose CL-6B spin columns (Pharmacia, Uppsala, Sweden) in VSB. Murine albumin at 1 mg/ml and 10% glycerol were added to gel-filtered virus stock and immediately frozen in aliquots. The viral concentration was initially estimated spectrophotometrically by absorbance at 260 nm, and the infectious titer of all viral stocks was determined by plaque assay on 293 cells using standard techniques [20,21].

2.2 Animals
Crossbred swine (50 kg) were obtained from Walnut Hill Farms (Hillsborough, NC, USA), housed under standard conditions and fed a regular diet. The Animal Care and Use Committee of Duke University approved all procedures and protocols. Animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 85-23, revised 1985).

2.3 TMR and in vivo adenoviral-mediated gene transfer
A total of 20 swine were used for the study. Fourteen animals comprised the experimental group and underwent TMR plus injection of Ad.Pac β-gal in the anterolateral left ventricle with Ad.Pac β-gal injection alone in the posterolateral left ventricle. The positive control group consisted of six animals. Three underwent TMR alone in the anterolateral left ventricle with vehicle injection alone in the posterolateral left ventricle. An additional three underwent injection of Ad.null alone in the anterolateral left ventricle with no treatment of the posterolateral ventricle.

All animals underwent induction of anesthesia with ketamine (22 mg/kg i.m.) and diazepam (10 mg i.v.). Orotracheal intubation was performed and anesthesia maintained with isoflurane (2–3%) while the animals were mechanically ventilated. Continuous electrocardiographic and pulse-oximetric monitoring was used throughout the procedure to ensure a stable cardiac rhythm and adequate oxygenation. Cefazolin (1 g i.v.) and bretylium tosylate (5 mg/kg i.v.) were given preoperatively. For the experimental group animals, a left anterolateral thoracotomy was performed through the fourth intercostal space under sterile conditions. The heart was suspended in a pericardial cradle and TMR performed using a pulsed solid-state holmium:YAG laser (Eclipse Surgical Technologies, Sunnyvale, CA, USA) to create five channels at 2-cm intervals in the anterolateral free wall of the left ventricle. This 2-cm interval was chosen to insure that gene product detected in and around a given TMR channel was due to the aliquot of vector injected into that channel and did not result from diffusion following injection of a neighboring channel [22]. Care was taken to avoid all epicardial and intramyocardial vessels. Channels were created using multiple 1.6 J pulses, with a total energy level of approximately 14.4 J per channel. Transmural penetration of laser channels was confirmed by visible spurting of blood from the channels during systole as well a change in the pitch of the sound emitted by the laser as it passed through the wall and into the blood filled ventricle. Hemostasis was obtained by manual compression.

After TMR was complete and hemostasis obtained, 1x109 plaque-forming units (pfu) of Ad.Pac β-gal were injected into each of the five channels at mid-myocardial level using a prototype device designed to facilitate precise intramyocardial injection (Microheart, Eldorado Hills, CA, USA). This device delivers exactly 100 µl of injectate via a 27-gauge needle attached to a stabilizer, which regulates the depth of injection. Up to ten injections are possible per device load. The location of each channel was marked with a loosely tied 6-0 polypropylene epicardial suture. An additional five identical direct intramyocardial injections of Ad.Pac β-gal were made at 2-cm intervals in the postlateral wall of the left ventricle without preceding TMR. These were marked with polypropylene suture as well. The high-titer viral stock was maintained on dry ice and thawed and diluted to the titer of approximately 1x109 pfu/0.1 ml with serum-free DMEM immediately before use. The pericardium was left widely open. A 20 French chest tube was placed and the wound closed in layers. The chest tube was removed at the conclusion of the procedure after the animal no longer required positive pressure mechanical ventilation.

Control animals underwent induction of anesthesia and thoracotomy as described for the experimental group. In three animals, TMR was performed as above to create five channels in the anterolateral left ventricular free wall. Adjuvant gene transfer was not performed. In the posterolateral left ventricle of these same animals, five direct injections of vehicle were performed. In the other three control animals, five injections of 1x109 pfu Ad.null were made as described in the anterolateral left ventricle, while no treatment was applied to the posterolateral free wall.

2.4 Analysis of gene transfer
Experimental animals were sacrificed under general anesthesia as described above at either 3 (n=6), 7 (n=6), or 14 days (n=2) postoperatively. Control TMR alone/vehicle (n=3) and empty virus/naïve (n=3) animals were sacrificed at 3 days postoperatively. These time points were chosen because of prior work demonstrating that foreign gene expression by adenoviral vectors generally peaks within 7 days [16,17,19,20]. The beating hearts were removed via redo-left thoracotomy. The regions of TMR plus direct Ad.Pac β-gal injection and direct Ad.Pac β-gal injection alone from the experimental animals, as well as the TMR, vehicle, and Ad.null alone regions from the positive controls, were identified by the location of the epicardial sutures. Immediately following sacrifice, each region was excised, blotted dry, and cut to a weight of 2.0±0.1 g. Four of the five regions were randomly chosen for quantitation of β-galactosidase protein levels and the remaining region was used for histologic analysis. The regions chosen for determination of protein levels were immediately frozen at –80°C for protein extraction, and the remaining region processed for frozen sections and immunohistochemistry. The regions randomly chosen for histologic analysis were equilibrated in 30% sucrose in phosphate buffered saline (PBS) at 4°C, then embedded in both longitudinal and cross-section in OCT compound (Optimal Cutting Temperature, Sakura Finetek USA, Torrance, CA, USA) and snap frozen in liquid nitrogen. Frozen sections (6 µm) were made in a cryostat on microscope slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA, USA). Slides were allowed to come to room temperature. They were then fixed in 10% formalin for 2 min followed by three 5-min washes in PBS. Fixed tissue was then stained for β-galactosidase by incubating in X-gal solution [20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 containing 0.5 mg/ml X-gal (3-chloro-5-bromo-indolyl-β-galactopyranoside)] for at least 90 min at 37°C. Three additional 5 min washes in PBS were then performed and the tissue sections fixed in 10% formalin overnight. Sections (5 µm) were then counterstained with eosin.

β-Galactosidase protein concentration was quantified using an enzyme-linked immunosorbent assay (ELISA) (3 Prime-5 Prime, Boulder, CO, USA) according to the manufacturer’s instructions. Frozen tissue sections were thawed and sonicated on ice in lysis buffer [50 mM Tris, pH 7.4, 1 mM EDTA, 0.1% Triton-X100 containing phenylmethyl-sulfonylfluoride (0.2 mg/ml) and leupeptin (0.5 µg/ml)]. Tissue debris was pelleted by centrifugation at 14 000 g and total protein concentration was measured using the Bradford assay (Bio-Rad, Richmond, CA, USA). β-Galactosidase protein quantity was determined as pg β-galactosidase/g tissue protein.

In the experimental animals, a section of non-injected septum was taken from each heart and prepared for ELISA as described above and served as a negative control. To assess for the systemic distribution of adenovirus, sections of spleen and liver were excised and prepared as described above as well.

2.5 Immunohistochemistry
To characterize and compare the inflammatory response in the direct viral injection versus TMR plus direct injection regions, as well as in the control TMR and vehicle regions, tissue sections randomly selected for histologic analysis were also used for immunochemistry to identify leukocytes with primary antibodies directed against CD-18, CD-4, and CD-8 (Serotec, Oxford, UK). The CD-18 antibody recognizes the β2 chain of the leukocyte-adherence glycoprotein complex [23,24]. This integrin is present on activated monocytes and neutrophils and mediates endothelial adhesion and migration [25]. CD-4 and CD-8 are found on T-helper and cytotoxic T-cells, respectively [26]. These latter cells have been demonstrated to be involved in the cell mediated immunity limiting the duration of transgene expression following adenoviral-mediated gene transfer [27–29]. Sections were embedded as described above. Frozen sections were thawed, acetone fixed, and dried at room temperature and then equilibrated in PBS. Blocking solution (1.5% horse serum in PBS) was applied for 1 h at room temperature. Antibodies were diluted in PBS at 1:10 concentration and were applied to tissue sections for 1 h. This was followed by sequential incubation with biotinylated anti-mouse IgG and ABC reagent according the manufacturer’s specifications (Vectastain ABC kit, Vector Laboratories). Levamisole was added to block endogenous alkaline phosphatase activity, and immune complexes were localized using the chromogenic alkaline phosphatase substrate, Vector Red (Vector Laboratories). The sections were lightly counterstained with hematoxylin, dehydrated, and mounted with Permount (Fisher Scientific). In all experiments, an adjacent section was incubated with an irrelevant murine IgG monoclonal antibody to serve as a negative control. For the CD-4 and CD-8 assays, a section of spleen was used as a positive control. Staining intensity was quantified using an image analysis system (Olympus IX 70 inverted microscope, Optronics DEI-750 image-capturing hardware: PowerTowerPro 180 CPU). Images were captured using ADOBE PREMIERE and quantified using NIH IMAGE software.

2.6 Statistical analysis
Data was analyzed using STATISTICA for windows version 5.1 (StatSoft, Tulsa, OK, USA). All data is presented as the mean±standard error. Absolute values of transgene expression obtained by ELISA were compared for lased and non-lased regions within the same heart using a paired Student’s t-test. Values of transgene expression at 3 versus 7 days postoperatively in each region were compared using an unpaired Student’s t-test. Values of immune marker staining intensity were compared for lased, non-lased, and control regions using a one-way analysis of variance (ANOVA). Statistical significance was considered a P value of <0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
All animals survived to their predetermined times of sacrifice. The procedure was well tolerated in all instances, with food intake and activity levels returning to normal by 2 days postoperatively.

3.1 ELISA
A total of 24 direct Ad.Pac β-gal injection and 24 TMR plus Ad.Pac β-gal injection regions were analyzed for β-galactosidase protein concentration at both 3 and 7 days postoperatively. A total of eight direct inject and eight TMR plus direct inject regions were analyzed at 14 days postoperatively. ELISA data for transgene expression 3 and 7 days postoperatively are shown in Table 1. ELISA revealed significantly greater transgene expression in the direct injection as compared to the TMR plus direct inject regions 3 days postoperatively. These same results were seen 7 days postoperatively (Fig. 1). The was no significant difference in the degree of β-galactosidase expression seen at 3 versus 7 days postoperatively for the direct inject regions (P=0.3), although a significant decrease in transgene expression was seen at 7 versus 3 days in the TMR plus direct inject regions (P=0.03). No transgene expression was seen 14 days postoperatively in either the direct Ad.Pac β-gal injection or TMR plus Ad.Pac β-gal injection regions. Likewise, essentially no transgene expression (<1 pg β-gal/g protein) was detected in the positive control TMR alone, Ad.null, or naïve regions.


View this table:
[in this window]
[in a new window]

  		Table 1 Transgene expression as detected by ELISAa 3 and 7 days postoperatively

 

Figure 1
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Graph of transgene expression by ELISA in myocardial regions treated with TMR plus direct Ad.Pac β-gal injection and direct Ad.Pac β-gal injection alone 3, 7 and 14 days postoperatively. Note the significantly greater expression in direct inject regions both 3 and 7 days postoperatively. No transgene expression was detected in either the TMR plus direct viral injection or direct viral injection alone regions at 14 days postoperatively. Likewise, no transgene expression was detected in TMR alone, Ad.null, or naïve regions in the control animals.

 
ELISA revealed very little systemic distribution of the adenoviral vector, with mean values of 21.7±6.1 pg β-gal/g protein in the liver and 19.4±4.8 pg β-gal/g protein in the spleen 3 days postoperatively. Likewise, values in the non-injected septum were 4.9±1.5 pg β-gal/g protein at 3 days postoperatively.

3.2 Histology
β-Galactosidase staining of the direct Ad.Pac β-gal inject regions both 3 and 7 days following gene transfer revealed intensely blue staining cardiomyocytes at the site of injection (Fig. 2a). Histology of the TMR plus direct inject regions also revealed blue staining of cardiomyocytes in the vicinity of the laser channels (Fig. 2b), however the degree of staining was diminished compared to the direct inject alone regions. Higher power views demonstrate the X-gal staining to be localized to the nuclei of cardiomyocytes (Figs. 2c and d). No β-galactosidase staining was detected in the positive control TMR alone, Ad.null, or naïve regions.


Figure 2
View larger version (137K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Photomicrograph (original magnification 40x) demonstrating β-galactosidase activity in (A) non-lased and (B) lased myocardium 3 days following adenoviral gene transfer. The blue staining identifies areas of functional Ad.Pac β-gal transfection and expression. Note the large confluent areas of blue staining within cardiomyocytes in non-lased regions. This is contrasted with the relatively patchy staining noted in cardiomyocytes located at the periphery of the TMR channels (*denotes location of channel). Higher power views (original magnification 200x) confirm the β-galactosidase staining to be primarily localised to the nuclei 1 of cardiomyocytes in both the direct injection (C) and TMR plus direct injection (D) regions.

 
3.3 Immunohistochemistry
To assess the degree of inflammation associated with vehicle, TMR, and direct intramyocardial Ad.Pac β-gal injection alone, as well as TMR plus direct Ad.Pac β-gal injection, immunohistochemistry was used to identify CD-18+ leukocytes (Fig. 3) and CD-4+ and CD-8+ lymphocytes. CD-18 is a marker of activated monocytes and neutrophils [24,25], whereas CD-4 and CD-8 denote T-helper and cytotoxic T-cells [26] involved in cell mediated immunity limiting the duration of transgene expression following adenoviral-mediated gene transfer [27–29]. Quantitative analysis revealed significantly greater CD-18+ staining in the TMR plus direct viral injection (2.8±0.1 CD-18+ staining units) and TMR alone (2.0±0.2 CD-18+ staining units) regions as compared to direct viral injection alone (1.0±0.2 CD-18+ staining units) (both P<0.001) (Fig. 4). There was minimal CD-18+ staining following injection of vehicle alone. Transgene expression was greatest in areas distant from the inflammatory infiltrate. Three days postoperatively, there were only rare CD-4+ and CD-8+ cells detected in the TMR plus direct Ad.Pac β-gal inject, direct Ad.Pac β-gal inject alone, TMR alone, and vehicle treated regions (Fig. 5) suggesting that T-cells did not play a large role in the observed immune response. Increased numbers of CD-4+ and CD-8+ lymphocytes were seen at 7 days postoperatively (Fig. 5), although these cells still represented a minority of the observed inflammatory infiltrate.


Figure 3
View larger version (111K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Immunohistochemistry (original magnification 40x) of (A) non-lased and (B) lased myocardium 3 days following β-galactosidase adenoviral gene transfer. In (A) and (B) sections were first stained with X-gal to demonstrate transduced cells (blue nuclei) and then immunostained for CD-18+ leukocytes (CD-18+ leukocytes stain red). Note the greater inflammation in the TMR plus direct Ad.Pac β-gal injection regions as compared to direct Ad.Pac β-gal injection alone (*denotes location of TMR channel). Higher power views (original magnification 200x) demonstrate that essentially all of the cellular infiltrate consists of red staining CD-18+ leukocytes in both the direct inject alone (C) and TMR plus direct inject (D) regions.

 

Figure 4
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Graph of CD-18+ staining in myocardial regions treated with TMR plus direct Ad.Pac β-gal injection and direct Ad.Pac β-gal injection alone, as well as positive control TMR and vehicle alone, 3 days postoperatively. Note the significantly greater CD-18+ staining intensity, indicating increased inflammatory infiltrate, in the myocardial regions treated with TMR plus direct viral injection and TMR alone versus direct viral injection. There was minimal inflammation noted following vehicle alone.

 

Figure 5
View larger version (139K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Immunohistochemistry (original magnification 200x) staining for (A) CD-4+ and (B) CD-8+ lymphocytes (CD-4+ and 8+ lymphocytes stain red) 3 days following TMR plus β-galactosidase adenoviral gene transfer. Note rare CD-4+ and CD-8+ cells (arrows) interspersed among leukocyte infiltrate 3 days postoperatively. Increased numbers of both CD-4+ (C) and CD-8+ (D) lymphocytes are noted 7 days postoperatively, although still representing a minority of the inflammatory infiltrate.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
This study demonstrates that adenoviral-mediated gene transfer performed in conjunction with transmyocardial laser revascularization is possible, although the efficiency of transgene expression in regions of the heart treated with TMR was less and more transient than that seen following direct intramyocardial injection alone. Histochemical analysis revealed blue staining indicative of β-gal expression primarily in a limited number of cardiomyocytes in the vicinity of the TMR channels, as opposed to the more uniform staining seen following direct injection in non-lased regions. These findings suggest potential limitations in the clinical application of adenoviral-mediated gene delivery in conjunction with TMR. However, the study does demonstrate that the combination of the two therapies appears to be safe, as all animals tolerated the procedure well. Finally, consistent with prior work [22], there appears to be little systemic distribution of the transgene following direct intramyocardial application either with or without preceding TMR.

Previous work has demonstrated that an immune response against replication-defective adenoviral vectors is the major cause of transient transgene expression [27–31], and the diminished gene expression in TMR treated myocardium likely results from the immune response to laser injury, which may lead to a more intense attack on the virus than normally occurs. This is supported by the finding of an almost 3-fold increase in CD-18+ staining in the TMR plus direct Ad.Pac β-gal inject regions as compared to direct Ad.Pac β-gal injection alone. In addition, the greatest gene expression was observed distant from the inflammation, suggesting that the inflammatory response to the myocardial injury following TMR was responsible for the limited gene expression. That CD-18-positive TMR-mediated inflammation is the cause of limited transgene expression is supported by the finding of little to no CD-4+ or CD-8+ lymphocytes in the regions of inflammatory infiltrate 3 days postoperatively. Slightly increased numbers of these cells were noted 7 days postoperatively, yet still represented a minority of the inflammatory infiltrate. Because CD-4+ and CD-8+ lymphocytes are the primary mediators of specific virus-mediated immunity [27–29], their absence suggests that limited transgene expression in the setting of combined TMR and adenoviral-mediated gene transfer is due to myocardial injury from laser treatment and not from a specific immune response against the viral vectors. These findings are consistent with prior studies [32–36] describing the presence of an inflammatory infiltrate in the region of TMR channels during the first postoperative week. Because adenoviral DNA remains episomal and does not become incorporated into the host genome [17,37], the transgene may become diminished or lost during the period of rapid cell turnover associated with the healing process.

The inflammatory response to myocardial injury with the laser is not unlike that occurring following myocardial infarction, albeit on a smaller scale [32]. Interestingly, Leor et al. [37] have noted transgene expression to be significantly lower in infarcted as compared to non-infarcted myocardium following injection with an adenoviral vector encoding the reporter gene β-galactosidase. β-Gal expression was limited to viable cardiomyocytes at the periphery of the infarct, not unlike the finding of β-gal expression in myocytes surrounding the TMR channels in the present study. Leor et al. found transgene efficiency to be lowest at the time of peak inflammatory response and hypothesized that the diminished expression in infarcted myocardium was due to an activated immune response.

Despite the relatively low efficiency of transgene expression in lased myocardium, the ability to introduce genes even transiently into cardiomyocytes at the periphery of the TMR channels may be of therapeutic benefit. For example, Giordano et al. [11] demonstrated improved perfusion and function in ischemic myocardium following intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor-5. The functional improvements were still evident 12 weeks following gene transfer at a time when the episomal transgene would no longer be present. Thus, the beneficial effect of transgenes encoding angiogenic growth factors may persist long after transgene expression has waned [38].

To our knowledge, only one prior study has attempted to examine the interaction between TMR and adenoviral-mediated gene transfer. Following TMR, Fleischer et al. [36] administered an adenoviral vector carrying the cDNA for human profilin-I, a protein important for endothelial cell migration and adhesion. They were unable to document successful transfection at 28 days post-injection, consistent with the results of the present study. They also found no increase in neovascularization in regions treated with TMR plus the profilin carrying adenovirus as compared to those receiving TMR alone. This suggests that TMR may limit the therapeutic effectiveness of adjunctive adenoviral-mediated gene transfer. Of note, consistent with the present study, they found a significant increase in inflammation, as assessed qualitatively using routine histologic staining, in myocardial regions treated with adenovirus plus TMR versus TMR alone.

Limitations of the present study are several. First, the study was carried out in normal as opposed to chronically ischemic hearts, the condition under which TMR is applied clinically. However, histologic findings, including the degree of inflammation following TMR are similar in normal and chronically ischemic myocardium [35], suggesting the differences in transgene expression observed in the present study might also be expected to occur in chronically ischemic hearts. Second, the adenoviral vector studied contained the reporter-gene β-galactosidase rather than a functional gene encoding an angiogenic growth factor. However, β-gal allowed for easy detection and quantitation of transgene expression in accordance with the aims of the study. Finally, we chose to inject the adenoviral vector directly into the TMR channel once hemostasis was obtained. However, whether better gene expression might be obtained by injecting at some distance from the channel (and the associated inflammatory infiltrate) is unknown. Previous work has shown that laser-induced thermal damage following TMR may extend several millimeters from the channel [39]. Consequently, gene transfer may still be limited by vector injection into myocardial regions between the channels, especially given the fact that in clinical practice the channels are placed only 1 cm apart.

In summary, adenoviral-mediated gene transfer performed in conjunction with transmyocardial laser revascularization is feasible, although the efficiency of transgene expression is limited, apparently due to the augmented inflammatory response following laser injury of the myocardium. Transfection appears to be limited to a small number of cardiomyocytes at the periphery of the TMR channels. Despite this, the combination of TMR with adenoviral-mediated transfer of genes encoding for angiogenic growth factors represents an exciting new means to potentially augment therapeutic angiogenesis following TMR.

Time for primary review 25 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This work was supported in part through a National Research Service Award from the National Institutes of Health and the National Heart, Lung, and Blood Institute (G.C.H.) (Grant no. 1 F32 HL09969-01) as well as an unrestricted educational grant from Microheart, Inc. (Eldorado Hills, CA, USA) and Eclipse Surgical Technologies, Inc. (Sunnyvale, CA, USA). In addition, the authors gratefully acknowledge Mr. Michael Lowe for his surgical technical assistance.


   Notes
 
{star} Presented in part at the American College of Cardiology 48th Annual Scientific Session, March 7–10, 1999, New Orleans, Louisiana, USA. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

  1. Frazier O.H., Cooley D.A., Kadipasaoglu K.A., et al. Myocardial revascularization with laser. Preliminary findings. Circulation (1995) 92(suppl_II):II-58–II-65.[Medline]
  2. Cooley D.A., Frazier O.H., Kadipasaoglu K.A., et al. Transmyocardial laser revascularization: clinical experience with twelve-month follow-up. J Thorac Cardiovasc Surg (1996) 111:791–799.[Abstract/Free Full Text]
  3. Horvath K.A., Mannting F., Cummings N., Shernan S.K., Cohn L.H. Transmyocardial laser revascularization: operative techniques and clinical results at two years. J Thorac Cardiovasc Surg (1996) 111:1047–1053.[Abstract/Free Full Text]
  4. Horvath K.A., Cohn L.H., Cooley D.A., et al. Transmyocardial laser revascularization: results of a multicenter trial with transmyocardial laser revascularization used as sole therapy for end-stage coronary artery disease. J Thorac Cardiovasc Surg (1997) 113:645–654.[Abstract/Free Full Text]
  5. Donovan C.L., Landolfo K.P., Lowe J.E., Clements F., Coleman R.B., Ryan T. Improvement in inducible ischemia during dobutamine stress echocardiography after transmyocardial laser revascularization in patients with refractory angina pectoris. J Am Coll Cardiol (1997) 30:607–612.[Abstract]
  6. Hughes G.C., Lowe J.E., Kypson A.P., et al. Neovascularization after transmyocardial laser revascularization in a model of chronic ischemia. Ann Thorac Surg (1998) 66:2029–2036.[Abstract/Free Full Text]
  7. Milano A., Pratali S., Tartarini G., et al. Early results of transmyocardial revascularization with a holmium laser. Ann Thorac Surg (1998) 65:700–704.[Abstract/Free Full Text]
  8. Diegeler A., Schneider J., Lauer B., Mohr F.W., Kluge R. Transmyocardial laser revascularization using the holmium-YAG laser for treatment of end stage coronary artery disease. Eur J Cardiothorac Surg (1998) 13:392–397.[CrossRef][Web of Science][Medline]
  9. Schofield P.M., Sharples L.D., Caine N., et al. Transmyocardial laser revascularization in patients with refractory angina: a randomised controlled trial. Lancet (1999) 353:519–524.[CrossRef][Web of Science][Medline]
  10. Hughes G.C., Kypson A.P., St. Louis J.D., et al. Improved perfusion and contractile reserve after transmyocardial laser revascularization in a model of hibernating myocardium. Ann Thorac Surg (1999) 67:1714–1720.[Abstract/Free Full Text]
  11. Giordano F.J., Ping P., McKirnan M.D., et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med (1996) 2:534–539.[CrossRef][Web of Science][Medline]
  12. Mack C.A., Patel S.R., Schwarz E.A., et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg (1998) 1:168–176.
  13. Schumacher B., Pecher P., von Specht B.U., Stegmann Th. Induction of neoangiogenesis in ischemic myocardium by human growth factors. First clinical results of a new treatment of coronary heart disease. Circulation (1998) 97:645–650.[Abstract/Free Full Text]
  14. Isner J.M., Pieczek A., Schainfeld R., et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet (1996) 348:370–374.[CrossRef][Web of Science][Medline]
  15. Stewart M.J., Plautz G.E., Del Buono L., et al. Gene transfer in vivo with DNA-liposome complexes: safety and acute toxicity in mice. Hum Gene Ther (1992) 3:267–275.[Web of Science][Medline]
  16. Mühlhauser J., Merrill M.J., Pili R., et al. VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo. Circ Res (1995) 77:1077–1086.[Abstract/Free Full Text]
  17. Li K., Buvoli M., Welikson R.E., Vikstrom K.L., Leinwand L.A. Advances in cardiovascular gene transfer. Cardiologia (1997) 42:39–46.[Medline]
  18. Lazarous D.F., Shou M., Stiber J.A., et al. Pharmacodynamics of basic fibroblast growth factor: route of administration determines myocardial and systemic distribution. Cardiovasc Res (1997) 36:78–85.[Abstract/Free Full Text]
  19. Channon K.M., Blazing M.A., Shetty G.A., Potts K.E., George S.E. Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells. Cardiovasc Res (1996) 32:962–972.[Abstract/Free Full Text]
  20. Channon K.M., Fulton G.J., Gray J.L., et al. Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels. Cardiovasc Res (1997) 35:505–513.[Abstract/Free Full Text]
  21. Graham F.L., Prevec L. Gene transfer and expression protocols. (1991) Vol. 7. Clifton, NJ: Humana Press. 109–128.
  22. Magovern C.J., Mack C.A., Zhang J., et al. Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector. Ann Thorac Surg (1996) 62:425–434.[Abstract/Free Full Text]
  23. Galea-Lauri J., Blackford J., Wilkinson J.M. The expression of CD11/CD18 molecules on rabbit leukocytes: identification of monoclonal antibodies to CD18 and their effect on cellular adhesion processes. Mol Immunol (1993) 30:529–537.[CrossRef][Web of Science][Medline]
  24. Channon K.M., Fulton G.J., Davies M.G., et al. Modulation of tissue factor protein expression in experimental venous bypass grafts. Arterioscler Thromb Vasc Biol (1997) 17:1313–1319.[Abstract/Free Full Text]
  25. Smith C.W., Rothlein R., Hughes B.J., et al. Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration. J Clin Invest (1988) 82:1746–1756.[Web of Science][Medline]
  26. Abbas A.K., Lichtman A.H., Pober J.S. Cellular and molecular immunology. (1991) Philadelphia, PA: W.B. Saunders.
  27. French B.A., Mazur W., Geske R.S., Bolli R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors. Circulation (1994) 90:2414–2424.[Abstract/Free Full Text]
  28. French B.A., Mazur W., Ali J.M., et al. Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries, and two models of coronary restenosis. Circulation (1994) 90:2402–2413.[Abstract/Free Full Text]
  29. Quiñones M.J., Leor J., Kloner R.A., et al. Avoidance of immune response prolongs expression of genes delivered to the adult rat myocardium by replication-defective adenovirus. Circulation (1996) 94:1394–1401.[Abstract/Free Full Text]
  30. Guzman R.J., Lemarchand P., Crystal R.G., Epstein S.E., Finkel T. Efficient gene transfer into myocardium by direct injection of adenovirus vectors. Circ Res (1993) 73:1202–1207.[Abstract/Free Full Text]
  31. Gilgenkrantz H., Duboc D., Juillard V., et al. Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response. Hum Gene Ther (1995) 6:1265–1274.[Web of Science][Medline]
  32. Hardy R.I., Bove K.E., James F.W., Kaplan S., Goldman L. A histologic study of laser-induced transmyocardial channels. Lasers Surg Med (1987) 6:563–573.[Web of Science][Medline]
  33. Krabatsch T., Schaper F., Leder C., Tulsner J., Thalmann U., Hetzer R. Histological findings after transmyocardial laser revascularization. J Card Surg (1996) 11:326–331.[Web of Science][Medline]
  34. Gassler N., Wintzer H.O., Stubbe H.M., Wullbrand A., Helmchen U. Transmyocardial laser revascularization. Histological features in human nonresponder myocardium. Circulation (1997) 95:371–375.[Abstract/Free Full Text]
  35. Fisher P.E., Khomoto T., DeRosa C.M., Spotnitz H.M., Smith C.R., Burkhoff D. Histologic analysis of transmyocardial channels: comparison of CO2 and holmium:YAG lasers. Ann Thorac Surg (1997) 64:466–472.[Abstract/Free Full Text]
  36. Fleischer K.J., Goldschmidt-Clermont P.J., Fonger J.D., et al. One-month histologic response of transmyocardial laser channels with molecular intervention. Ann Thorac Surg (1996) 62:1051–1058.[Abstract/Free Full Text]
  37. Leor J., Quiñones M.J., Patterson M., Kedes L., Kloner R.A. Adenovirus-mediated gene transfer into infarcted myocardium: feasibility, timing, and location of expression. J Mol Cell Cardiol (1996) 28:2057–2067.[CrossRef][Web of Science][Medline]
  38. Lee J.S., Feldman A.M. Gene therapy for therapeutic myocardial angiogenesis: a promising synthesis of two emerging technologies. Nat Med (1998) 4:739–742.[CrossRef][Web of Science][Medline]
  39. Jansen E.D., Frenz M., Kadipasaoglu K.A., et al. Laser-tissue interaction during transmyocardial laser revascularization. Ann Thorac Surg (1997) 63:640–647.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Ann. Thorac. Surg.Home page
T. Asai, S. Yamamoto, K. Ishino, T. Kohmoto, M. Kuriyama, G. Kato, Y. Oshima, N. Yamamoto, K. Notohara, S. Okada, et al.
Time-Dependent Regional Myocardial Denervation as a Nonspecific Response to Transmyocardial Laser Revascularization
Ann. Thorac. Surg., October 1, 2005; 80(4): 1362 - 1369.
[Abstract] [Full Text] [PDF]

Home page
 Card Surg AdultHome page
M. Ruel, R. A. Kelly, and F. W. Sellke
Therapeutic Angiogenesis, Transmyocardial Laser Revascularization, and Cell Therapy
Card. Surg. Adult, January 1, 2003; 2(2003): 715 - 750.
[Full Text]

Home page
 J Am Coll CardiolHome page
G. C. Hughes, S. S. Biswas, B. Yin, D. V. Baklanov, B. H. Annex, R. E. Coleman, T. R. DeGrado, C. K. Landolfo, K. P. Landolfo, and J. E. Lowe
A comparison of mechanical and laser transmyocardial revascularization for induction of angiogenesis and arteriogenesis in chronically ischemic myocardium
J. Am. Coll. Cardiol., April 3, 2002; 39(7): 1220 - 1228.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Hughes, G.C.
Right arrow Articles by Landolfo, K. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hughes, G.C.
Right arrow Articles by Landolfo, K. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?