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Eur J Cardiothorac Surg 2003;23:620-625
© 2003 Elsevier Science NL

Gene expression profiling of human stenotic aorto-coronary bypass grafts by cDNA array analysis

^a Department of Thoracic and Cardiovascular Surgery, Johannes Gutenberg University, Mainz, Germany
^b Department of Internal Medicine, Johannes Gutenberg University, Mainz, Germany
^c Department of Thoracic and Cardiovascular Surgery, University Regensburg, Regensburg, Germany
^d Department of Pathology, Johannes Gutenberg University, Mainz, Germany

Received 22 May 2002; received in revised form 21 December 2002; accepted 7 January 2003.

^* Corresponding author. Tel.: +49-6131-172106; fax: +49-6131-470193
e-mail: hilker{at}mail.uni-mainz.de

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objective: Aorto-coronary bypass graft disease with its increasing clinical signification represents an unsolved problem in cardiological and heart surgery practice. Late occlusion of autologous saphenous vein grafts is due to medial and neointimal thickening secondary to migration and proliferation of smooth muscle cells (SMCs) and the subsequent formation of atherosclerotic plaques. This study is aimed at identifying differentially expressed genes in human stenotic bypass grafts to detect unknown pathomechanism and to identify novel targets for prophylactic treatment options. Methods: Stenotic saphenous aorto-coronary bypass grafts (n=5) were retrieved during re-do aorto-coronary bypass surgery. Ungrafted saphenous vein segments (n=5) were taken from the same group of patients and served as internal controls. cDNA samples were prepared and hybridized to cDNA arrays. Results: Some of the differentially expressed genes complied with expected gene expression including upregulation of c-jun and CDK10. In addition, previously unidentified gene expression patterns were detected such as upregulation of HSP70, fibronectin1, erbB3 proto-oncogene and c-myc. To confirm the latter finding, upregulation of c-myc in neointimal and medial SMCs of stenotic graft segments was confirmed by in situ hybridization studies and by immunhistochemistry. Conclusion: Gene expression patterns of human stenotic bypass grafts retrieved by re-do operations can be reliably analyzed by cDNA array technology. With this technique, new therapeutic targets in patients could be identified as shown by the findings regarding c-myc. c-myc is a proto-oncogene acting as a transcription factor and blocking c-myc has shown a reduction of neointima formation in animal models. Our study yields a rational for the use of antisense c-myc oligonucleotides to reduce neointima formation and to avoid stenosis in patients.

Key Words: Bypass graft disease • cDNA array • c-myc • In situ hybridization

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
The pioneering work of Favaloro and the first vein graft implantation in a patient by Garett and co-workers established the era of surgical revascularization for the treatment of ischemic heart disease. The use of the saphenous vein as a bypass conduit rapidly gained widespread acceptance, as an effective treatment for angina. However, surgical revascularization has significant shortcomings, in particular, the high rate of accelerated atherosclerosis that develops in vein grafts: 10 years after surgery, 40% of grafts are occluded and only 50% are free of significant atherosclerotic disease [1].

With about 600,000 coronary bypass operations all over the World, restenosis-induced graft failure presents an unsolved clinical problem. In 90% of the patients with late bypass graft occlusion, neointima formation is responsible [2,3]. Neointima formation is considered an arterial healing response that is initiated by dedifferentiation of vascular smooth muscle cells (SMCs), followed by their emigration, proliferation and subsequent elaboration of abundant extracellular matrix [4–7]. Because of the limited availability of human stenotic saphenous bypass grafts for research purposes, our current understanding of neointima formation is based almost exclusively on animal models [8,9]. However, therapeutic concepts for the prevention of neointima formation derived from these animal models, so far have not substantially improved the clinical success of bypass grafting [10,11]. This suggests major differences in neointima formation between animals and humans. Accordingly, molecular studies on human tissue samples are required to develop novel treatment strategies. This study is meant to establish a method for profiling gene expression in human stenotic bypass grafts to identify differentially expressed genes that may serve as novel therapeutic targets. We applied differential gene expression screening using cDNA array technology in explanted bypass grafts, harvested during re-do surgery.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Patients and probe preparation
Our study group included five patients who were treated for recurrent angina and angiographically documented stenosis of the bypass grafts with re-do bypass surgery. Small segments of stenotic bypass and of native vein segments were snap frozen after surgical removal and stored at -80°C until analysis. The mean age of the patients was 66.7±8.3 years. Four patients were males, one female. The average period of time between graft implantation and explantation was 94.1 months. As isotype matched controls, we used small native vein segments, from the same patients, that were left over after saphenous vein segments were cut to exactly fit onto the external aspect of the heart during coronary bypass surgery. This has been approved by the local ethic committee.

2.2. RNA isolation
Total RNA was isolated using the guanidium thiocyanate phenol chloroform extraction method. RNA quantity was checked photometrically by absorption at 260 nm and quality was determined by examination of the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. After two phenol/chloroform extractions, RNA was precipitated with isopropanol, washed with 80% ethanol and air-dried. To remove genomic DNA contamination, RNA was treated with ribonuclease (RNase)-free deoxyribonuclease (DNase) I (Clontech, Palo Alto, CA), and was then dissolved in RNase-free H₂O and stored at -80°C until analysis.

2.3. cDNA probe preparation
For cDNA probe synthesis, 5 µg of DNase-treated total RNA combined with 1 µl of commercial dialysis solution (CDS) primer mix (Clontech, Palo Alto, CA) in a total volume of 3 µl were heated to 70°C for 2 min and were then incubated for 2 min. A mixture consisting of 2 µl of 5x first-strand cDNA reaction buffer, 0.5 µl of 100 mM dithiothreitol (DTT), 1 µl of 10x diethylnitrophenyl thiophosphate (dNTP) mix and 3.5 µl [-³²P] deoxyadenosine triphosphate (dATP, 3000 Ci/mmol, 10 µCi/µl) was added into the tube and heated at 50°C for 25 min. The reaction was stopped by adding 1 µl termination mix. The cDNA probe was purified with a spin column. Incorporation of ³²P into the probe was determined by counting in a liquid scintillation counter.

2.4. Hybridization and quantification of cDNA arrays
The Atlas human trial array containing cDNA fragments of 91 human genes/clones was purchased from Clontech. For the final essential experiments, only unstripped array membranes were used. These were prehybridized with 5 ml of ExpressHyb solution (Clontech, Palo Alto, CA) at 68°C with continuous rotation in a glass hybridization roller. After prehybridization for 2 h, purified -³²P-labeled cDNA probes derived from normal or stenotic vessel RNAs were added into different rollers, and hybridization was continued overnight at the same temperature. Arrays were subsequently washed twice in 200 ml of wash solution 1 (2x SSC, 1% sodium dodecyl sulfate (SDS)) at 68°C for 20 min with agitation and then washed once in 200 ml of wash solution 2 (0.1 SSC, 0.5% SDS) at 68°C for 20 min with agitation. After a final wash with 200 ml of 2x SSC for 5 min at room temperature, the damp membranes were sealed in plastic wrap and exposed to Kodak Biomax MS X-ray film with an intensifying screen at -80°C for 4 days.

Array images on the X-ray film were scanned at 400 dpi by using an image scanner and then analyzed using AtlasImage 2.0 software (Clontech, Palo Alto). We first eliminated false positive signals due to apparent artifacts by visual inspection; the intensity of each spot on the array was then calculated after background subtraction. For comparison between two arrays, the ‘global’ mode was used. Furthermore, the arrays obtained from one patient (control native vein and stenotic bypass) were compared. Putative functions of the genes identified were obtained by use of the AtlasInfo database4. To assess the reproducibility of this system, we repeated hybridization for three samples using new probes synthesized from the original total RNA. The majority (>90%) of expression signals were found reproducible. Wherever possible, we checked the RNA extraction method.

2.5. Statistical analysis
Data are expressed as average±SEM. Statistical significance was evaluated by Wilcoxon rank sum test to compare data in stenotic bypass grafts and in native saphenous veins using the Prism 3.0 software (GraphPad Software, San Diego, USA).

2.6. In situ hybridization
Specific riboprobes based on the coding region of the published human c-myc sequence were generated using the 3'RACE kit supplied by Gibco (Gibco BRL, Eggenstein, Germany) as described before. The first amplification was done with the universal amplification primer provided with the system and the c-myc-specific primer P1 as an upstream primer (P1=5'-CAAGAGGGTCAAGTTGGACAGTGTC-3'). The RACE product was reamplified with nested primers (upstream P2=5'-AACACAACGTCTTGGAGCGCCAG-3', downstream P3=5'-TGTTTTCCAACTCCGGGATCTGGTC-3'). All polymerase chain reactions (PCRs) were carried out with a DNA Thermal Cycler (TC9600, Perkin Elmer, Norwalk, USA) in a 50 µl assay composed of 2 U Taq Polymerase (Gibco BRL, Eggenstein, Germany) in 1xTaq buffer (Roche Diagnostics, Mannheim, German) containing 1.5 mM MgCl₂, 5% dimethyl sulfoxide (DMSO), 200 mM of each dNTP and 20 pmol of each primer. Cycling was done as follows: (1) long denaturation=94°C for 2 min; (2) 40 cycles with denaturation at 94°C for 15 s, annealing at 60°C for 15 s and extension at 72°C for 60 s; (3) The reaction was stopped by keeping the temperature at 72°C for 10 min followed by cooling down to 4°C. The second amplification yielded an 89 bp long fragment of the exon 3 c-myc mRNA, which was cloned into the pGEM-T vector system (Promega, Madison, USA). To ensure that the same portions of the multiple cloning sites were included in both the antisense and the sense probe, different clones with reverse orientations were isolated and checked by sequencing. The c-myc probe was screened using a GenBank (NCBI) to reduce the chance of cross-hybridization. After linearizing the plasmid-insert-templates with Spe I, ‘run-off’ RNA transcripts were generated by in vitro transcription using the T7 RNA promoter. The probes were labeled by incorporation of digoxigenin (DIG) linked uridine 5'-triphosphates (UTPs, DIG RNA labeling kit, Roche Diagnostics, Mannheim, Germany) following the manufacturer's instruction.

After paraffin removal and rehydration, 3 µm-thick tissue sections were digested with proteinase K (10 µg/ml) for 15 min at 37°C and washed with phosphate-buffered saline (PBS) containing 0.2% glycine and 0.1% Tween-20. Following pre-treatment, sections were dehydrated through a graded ethanol series. The hybridization reactions were done overnight with the in situ workstation (MWG Biotech, Ebersberg, Germany) at 41°C. After hybridization, the sections were consecutively washed with 2xSSC (2x10 min room temperature), 0.2xSSC (1x20 min room temperature) and 0.1xSSC (1x20 min, 55°C). The tissue was then rinsed at room temperature in P1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for 5 min. Blocking was done for 30 min at room temperature with the same buffer containing 1% blocking reagent (P2). Visualization of the labeled RNA:RNA-hybrids were carried out with an anti-DIG antibody conjugated with rhodamine (Roche Diagnostics, Mannheim, Germany; 1:4 in P2).

2.7. Immunhistochemistry
Formalin-fixed, paraffin-embedded sections of five stenotic bypass grafts analyzed in the cDNA expression array were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min at room temperature. The sections were microwaved in antigen unmasking solution (Biogenix, San Ramon, CA). The sections were incubated with the primary antibody directed against c-myc (clone 9E10, Biogenix, 1:50, in 1% blocking reagent), at 4°C. The secondary antibody was applied with the Super Sensitive Detection Kit (Biogenix, San Ramon, CA) following the manufacturer's instruction. The detection was carried out with 0.1% 3,3'-diaminobenzidine (DAB, Roche; Mannheim, Germany). Sections without primary antibody served as negative controls.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. Differential gene expression in stenotic bypass grafts
In order to improve our understanding of bypass graft disease, we used cDNA expression arrays for the simultaneous assessment of the expression of different genes. Using this approach, it was possible to identify differentially expressed genes (P<0.05) such as erbB3, HSP70, fibronectin1, c-myc, cyclin dependent kinase 10 (CDK10) and c-jun. Fig. 1 shows a representative array, hybridized to the cDNA of a native vein and to the cDNA of a stenotic bypass graft. The results and the putative functions of differentially expressed genes are summarized in Table 1. Among these, the c-myc mRNA level was consistently upregulated (5/5) which showed a 2.4±0.08-fold increase in c-myc mRNA expression in stenotic bypass grafts as compared to control veins. The CDK10, a cell cycle regulating kinase showed an average increase by a factor of 1.6±0.21, but this increase was not uniform: only three of five tested veins showed an enhanced expression of more than twofold. Downregulation of some genes were also seen but there were no statistical significance compared to native veins. Housekeeping genes consistently gave comparable positive results (Fig. 1).

View larger version (72K): [in this window] [in a new window]   Fig. 1. Representative cDNA array autoradiographs of native vein and stenotic aorto-coronary bypass graft. Spot 1 (erbB3), 2 (HSP70), 3 (fibronectin1), 4 (c-myc), 5 (CDK10) and 6 (c-jun) represent upregulated genes in stenotic bypass grafts. Housekeeping genes are spotted at the bottom lane (such as ubiquitin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, 60S ribosomal protein, 40S ribosomal protein).

View larger version (82K): [in this window] [in a new window]   Fig. 2. Histomorphological and in situ hybridization studies of stenotic bypass grafts vs. control veins. (A) Masson Goldner staining. Luminal narrowing caused by an extensive intimal thickening. (B–G) In situ hybridization for c-myc mRNA in stenotic bypass grafts and control veins using DIG-labeled antisense probes. Note the robust hybridization signal in neointimal SMCs of stenotic bypass grafts (D,G). In contrast, detection of c-myc mRNA in native veins was limited to the luminal endothelial layer (B) and to endothelial cells of vasa vasorum in the adventitia (F). (C and E) are the corresponding phase contrast images to (B and D). (H) Immunhistochemical staining using an monoclonal antibody against c-myc confirms the locoregional distribution pattern seen in the in situ hybridization studies.

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
To identify genetic alteration in stenosed human bypass grafts, we used the cDNA array technology to analyze the expression pattern in stenotic bypass grafts in comparison to native vein grafts. The principal findings of this study are the following two topics: (1) erbB3, HSP70, fibronectin 1, c-myc, jun and CDK10 are upregulated in stenotic bypass grafts. (2) We discovered previously unknown gene expression events such as upregulation of the proto-oncogene c-myc [12]. Furthermore, it was possible to confirm the array results by in situ hybridization and interestingly, also with positive immunhistochemistry. Although neointimal hyperplasia is the key mechanism in vein graft failure, the exact biological events involved in this pathologic processes (bypass graft disease) are poorly understood [3,13,14]. SMC proliferation and migration are the central events in this process, induced by specific peptide growth factors such as the platelet-derived growth factor (PDGF) or insulin-like growth factor [15,16]. Several of these factors have also been linked to increased expression of the immediate early growth response genes (IEGR) such as c-myc, c-fos and c-jun [17,18]. Especially, the proto-oncogene c-myc is considered to play an important role in regulating cellular proliferation, migration and differentiation and is also involved in functional modifications affecting protein synthesis and apoptosis [19–21]. c-myc is typically expressed to a minimal extent or may even be absent during quiescent periods of the cell cycle, and upregulation of c-myc expression has been documented during periods of active cell division. The expression of c-myc is not only limited to the G0/G1 interface, but it is continuously expressed throughout the cell cycle. Besides, the initiation of vascular SMC proliferation c-myc also stimulates cell cycle progression [22]. Therefore, it appears feasible that a directed inhibition of c-myc activity using antisense oligonucleotides complementary to c-myc mRNA not only suppress cell cycle entry, but also arrest proliferating cells [10]. That this may indeed be operative in vivo has been suggested in previous animal experiments [22–25].

It is to be pointed out that the cDNA array method examines the mRNA level, not the protein concentrations and that these can be regulated not only by transcriptional but also by posttranscriptional mechanisms. Therefore, posttranslational modification of proteins is also an important mode of regulation that cannot be detected by DNA arrays. Some genes with small changes in mRNA level (<twofold) but with significant change in protein level have not been identified in this study. It was possible to demonstrate that the change in c-myc mRNA expression was also associated with an upregulation of c-myc protein in stenotic bypass grafts (Fig. 2H).

In the present study, we have focused on the feasibility of gene expression profiling and on genes that are potentially involved in the mechanisms of bypass graft disease. We cannot exclude the possibility that the gene expression profiles of the control veins (native saphenous veins harvested before implantation in the arterial circulation) may not perfectly reflect gene expression in a normal vessel, presumably due to other pathological changes. A perfect comparison between stenotic bypass grafts and control cannot be achieved in a clinical study. The standard ‘normal vs. diseased’ type of comparison, being the basis of profiling studies, cannot be defined easily in this context. Gene expression in normal tissue is likely to be dependent on several factors involving variations between patients and samples. We have tried to limit this problem by comparing native veins and stenotic bypass grafts obtained from the same individual. A further limitation of expression profiling is that any given tissue is, of course, composed of several cell types. In the case of studies on vessel wall segments, this includes SMCs, fibroblasts, endothelial cells, red blood cells and many cells of the immune system. Cells from each of these populations will have different functions at various stages of development and levels of activation. The result is a highly heterogeneous sampling of cells, each of them expressing a special set of genes. An expression profiling generated from a microarray study of such a sample will, of course, represent a snapshot of genes expressed by a large number of different cells at the moment of harvesting. We have demonstrated that such limitations of gene profiling can be overcome by the subsequent confirmation in locoregional assays, such as in situ hybridization technique, to localize the specific transcripts at a single cell level.

In conclusion, the present study demonstrates that it is possible to identify differentially expressed genes in human stenotic bypass grafts using the gene array technology. An initial analysis of differentially expressed genes revealed several interesting candidates that may be involved in neointima formation during bypass graft disease. We believe that this approach will contribute to a better understanding of the molecular processes involved in bypass graft failure.

	Acknowledgments

 
This study was supported by a grant of the Robert–Müller Foundation. We thank Marion Schadek-Bätz for help with the preparation of the manuscript.

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Michael Hilker Ulrich Hake Wlodzimierz Kuroczynski Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hilker, M. Articles by Buerke, M. Search for Related Content PubMed PubMed Citation Articles by Hilker, M. Articles by Buerke, M. Related Collections Cardiac - other