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Cardiovascular Research 1999 44(2):436-448; doi:10.1016/S0008-6363(99)00220-5
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Alterations in c-fos expression, cell proliferation and apoptosis in pressure distended human saphenous vein

J Galea1,a, J Armstrong1, S.E Francisa,a, G Cooperb, D.C Crossmana and C.M Holta,*

aCardiovascular Medicine, University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK
bCardiothoracic Surgery, Northern General Hospital, Sheffield S5 7AU, UK

* Corresponding author. Tel.: +44-114-271-4973; fax: +44-114-261-9587 c.holt{at}sheffield.ac.uk

Received 9 June 1999; accepted 25 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Saphenous vein graft failures, resulting from thrombosis and the abnormal proliferation, migration and apoptosis of vascular smooth muscle cells (VSMC) are major limitations of coronary artery bypass surgery. We investigated whether surgical trauma of human saphenous vein induces the early response gene c-fos and causes alterations in rates of proliferation and apoptosis. Methods: Surgically prepared human vein consisted of distended (at 350 mmHg for 2 min) or non-distended segments of vein maintained in serum free RPMI at 37°C and 5% CO2 for various time intervals. c-fos expression was detected by Northern analysis. Cell proliferation and apoptosis were determined by [3H]thymidine incorporation combined with proliferating cell nuclear antigen (PCNA) immunostaining and TUNEL, respectively. Labelling indices for proliferation and apoptosis were correlated with vessel wall thicknesses. Results: Control, freshly isolated vein expressed no c-fos. Surgically prepared vein synthesized c-fos 1 h following harvesting. There was a significant increase in c-fos in distended compared to non-distended vein. c-Fos protein increased in surgically prepared vein 24 h after harvesting. There was a significant increase in vascular cell proliferation in the non-distended compared to the distended vein: mean (S.E.M.) 1279 (218) vs. 863 (155) dpm/µg DNA, P<0.05, n=6. In addition, the apoptotic index was significantly lower in the media of non-distended vs. distended vein 0.82 (0.2) vs. 5.5 (1.5), P<0.05, n=5. Conclusions: These findings demonstrate that surgical preparation of human saphenous vein increases expression of c-fos mRNA and apoptosis and reduces proliferation when compared with non-distended vein. These changes may influence the failure of saphenous vein grafts.

KEYWORDS Apoptosis; Cardiovascular surgery; Gene expression; Smooth muscle; Veins


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In spite of disappointing long term results, the saphenous vein (SV) is the graft of choice in peripheral vascular disease [1] and the most frequently used conduit in coronary artery bypass graft surgery [2]. Graft occlusion is a major problem associated with SV grafts. Early occlusion, within 30 days of surgery, results principally from intraluminal thrombosis and is presumed to arise from damage to the endothelium [3] occurring during surgical manipulation. Vein dissection, handling of the vein with surgical instruments and adventitial stripping are all potentially traumatic. Surgical distention of the vein with fluid (e.g. heparinised blood or saline) to check for leakage from side branches is known to injure both the endothelial and medial cell layers as demonstrated by several quantitative, morphological and biochemical studies [4–6]. Endothelial damage, due to mechanical trauma and wall stretching during luminal distention, leads to the adherence and activation of platelets and the expression of tissue factor resulting in thrombosis. Furthermore these events may contribute to the development of intimal hyperplasia, which is the result of the abnormal migration and proliferation of vascular smooth muscle cells [7,8], the deposition of extracellular matrix [9] and graft atherosclerosis with lipid deposition [10]. Such intimal hyperplasia is the cause of intermediate and late occlusion of saphenous vein grafts resulting in narrowing of the vessel lumen. It is possible that surgical preparation, in particular vessel distention, causes an upregulation in the level of expression of genes such as c-fos which may be important in initiating longer term responses and the subsequent development of myointimal hyperplasia. A number of growth factors are produced by the cells of the vessel wall and these may be capable of autocrine effects and early response genes may up regulate these factors [11,12]. By acting locally, such factors have the potential to play a central role in the development of myointimal hyperplasia. It has previously been demonstrated that the expression of PDGF B is induced in an experimental model of saphenous vein grafting [13]. Furthermore, it has recently been shown that the coagulation factor Xa stimulates the release of PDGF from vascular smooth muscle cells (VSMC) [14]. This finding suggests a link between the early events causing activation of the coagulation cascade and the later development of intimal hyperplasia that may involve PDGF.

Apoptosis has been identified as a feature of human vascular pathology, in particular restenotic lesions [15], primary atherosclerotic lesions [16,17], and human saphenous vein grafts [18,19]. It has also been shown to participate in the regulation of cellularity during intimal thickening in the rat aorta following balloon injury [20]. It is now clear that proto-oncogenes may signal cell proliferation and apoptosis, with the net balance determined by the growth factor environment [21]. AP-1, the hetero-dimer of c-fos and c-jun is well known to be associated with proliferation, but it also plays a role in apoptosis through association with a p53-dependent apoptotic pathway [22] The preparation of saphenous vein for grafting includes distention and storage and may provide signals responsible for upregulation of genes such as c-fos. The diverse action of c-fos on various coagulation and growth factor genes including PDGF may contribute to both the early and late failure of vein grafts via their effects on both the cell proliferation and death pathways. Increased levels of proto-oncogenes, including c-fos, have been shown to be induced early after balloon denudation in both a rabbit and a rat model [23,24]. The aims of the present study were to evaluate the effect of surgical preparation of human saphenous vein on the expression of the early response gene, c-fos and its product and to compare this with cell proliferation and apoptosis.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Isolation and preparation of saphenous vein
Segments of saphenous vein were obtained from 38 patients (two female); mean age: 60.4 years; age range 39–75 years, undergoing coronary artery bypass grafting. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3). The protocol was approved by the Hospital Ethics Committee and informed consent was obtained. Patients received their usual cardiovascular medication prior to surgery including nitrates, calcium channel blockers and β-blockers. Aspirin was stopped at least 7 days before surgery. A segment of freshly isolated vein (approximately 50 mm) was taken from the lower portion of the long saphenous vein using a ‘no touch’ technique [25] as soon after the first incision as possible and before the systemic administration of heparin sulphate. Control segments of vein were either snap frozen in liquid nitrogen in the operating theatre immediately after harvesting or fixed in 10% buffered formalin prior to processing and embedding in paraffin for histological examination. The remaining saphenous vein segments were then gently flushed until free of blood and transferred to the laboratory at room temperature (23°C) in HEPES-buffered RPMI-1640 culture medium (Life) supplemented with penicillin (100 µg/ml), streptomycin (100 Units/ml) and glutamine (2 mmol/l) (all from ICN Flow Labs, High Wycombe, UK). These segments of saphenous vein were then divided into distended and non-distended groups. Distention was for 2 min at 350–400 mmHg, chosen because such pressures have been shown to occur during the surgical preparation of the saphenous vein during coronary bypass surgery [26]. The non-distended segment of saphenous vein was maintained in medium for an equivalent period of time as the distended segment.

2.2 Maintenance of saphenous vein ex vivo
Intact segments of distended and non-distended saphenous vein were maintained in RPMI-1640 culture medium at 37°C, 5% CO2 for up to 24 h. Serum was excluded since this is known to induce the expression of c-fos and cellular proliferation. Following this specimens were snap frozen in liquid nitrogen or fixed in 10% buffered formalin solution.

2.3 Scanning electron microscopy
En face scanning electron microscopy was performed to determine the morphology of the endothelial surface of distended and non-distended saphenous vein. Vein segments were fixed in 3% v/v glutaraldehyde in sodium cacodylate buffer. Dehydrated specimens were then critical-point dried, sputter coated and observed on a scanning electron microscope (Philips 500).

2.4 Northern analysis
Early response gene expression was determined by Northern analysis using RNA extracted from a total of 22 patients with n=3 per time point. Frozen segments of saphenous vein were first pulverized under liquid nitrogen then total RNA was extracted using a modification of the method of Chomczynski et al. [27] using RNAzol (Biogenesis). RNA was then subjected to gel electrophoresis in 1.1% agarose—formaldehyde, transferred onto Hybond N (Amersham, UK) and crosslinked by exposure to ultraviolet light. Membranes were pre-hybridized in 5x SSPE, 0.1% SDS and 2x Denhardt's solution at 65°C then hybridized overnight with a random primed cDNA probe for c-fos. c-fos cDNA consisted of a 747-bp Acc1 fragment of c-fos–PGEM4Z (a kind gift from Dr Martin Nicklin, Section of Molecular Medicine, University of Sheffield).

Membranes were then washed with 2x SSC at 65°C for 15 min, followed by 2x SSC, 0.1% SDS for 30 min and a final wash in 0.1x SSC, 0.1% SDS to remove excess background. Membranes were then exposed to film (Hyperfilm β max, Amersham) at –80°C using double intensifying screens. Membranes were subsequently hybridized with 7B6 cDNA (a non-cycle dependent gene transcript) [28] to check RNA loading. All blots were quantified by densitometry and corrected for unequal loading with reference to 7B6.

2.5 Immunohistochemistry
c-Fos immunostaining was performed on transverse paraffin sections of saphenous vein on APES-coated slides. The sections were taken from control pieces of saphenous vein which were placed immediately into formalin on removal from the patient and from distended or non-distended veins maintained as described above at atmospheric intraluminal pressure for 24 h. Sections were de-waxed and rehydrated in xylene and graded alcohols, and endogenous peroxidase was inhibited with 3% hydrogen peroxide. Sections were blocked for 25 min with 10% goat serum in Tris-buffered saline (TBS) and incubated overnight at 4°C with two different polyclonal rabbit antibodies to c-Fos (a kind gift from Dr Gerard Evan, ICRF and Oncogene Research Products) diluted 1:1500 and 1:10, respectively, with TBS containing 10% goat serum. Sections were then washed three times in TBS containing 2% goat serum.

Goat biotinylated anti-rabbit antisera (Vector Laboratories Ltd., Peterborough, UK) was then applied followed by streptABComplex/HRP (Vector Labs Ltd., Peterborough, UK). Finally, antibody binding was visualized with diaminobenzidene (DAB) and sections were counterstained with Carazzi's haematoxylin. Positive controls consisted of serum stimulated ECV304 cells. Negative controls consisted of sections incubated with non-immune IgG at similar concentrations to the primary antibody, as well as sections without primary or secondary antibody.

2.6 In situ hybridization histochemistry
c-fos mRNA was detected and localized in situ on paraffin sections of saphenous vein. A single stranded antisense RNA probe, to exon 4 of human c-fos, was synthesized in the presence of [{alpha}-35S]UTP (Amersham International Plc.) and SP6 RNA polymerases using a commercially available transcription kit (Promega). Unincorporated nucleotides were removed using a spun Sephadex G50 column. A sense probe was also synthesized as a negative control. The probes were hydrolyzed into 200 nucleotide fragments by incubating with a carbonate hydrolysis buffer (4 mmol/l NaHCO3, 6 mmol/l Na2CO3) at 60°C for 40 min. The reaction was stopped with 3 M NaAc and 10% acetic acid, and the probe was precipitated with ethanol.

Sections were deparaffinised and rehydrated in graded alcohols, fixed in 4% paraformaldehyde in PBS and rinsed in 0.5x SSC. The sections were incubated with 1 µg/ml proteinase K for 7.5 min at 37°C, rinsed in 0.5x SSC and washed in 0.1 M triethanolamine, 0.9% NaCl and 0.25% acetic anhydride. Sections were then prehybridised in 100 µl hybridization buffer (50% formamide, 0.3 M NaCl, 20 mmol/l Tris, pH 8.0, 5 mmol/l EDTA, 1x Denhardt's, 10% dextran sulphate, 10 mmol/l dithiothreitol) in airtight boxes containing blotting paper soaked in 4x SSC and 50% formamide, at 55°C for 2 h.

The probes were diluted in hybridization buffer to allow 300 000 cpm per tissue section, and added to the prehybridised sections and hybridized overnight at 55°C. Slides were then rinsed in 2x SSC, 10 mmol/l beta-mercaptoethanol, 1 mmol/l EDTA and then immersed in RNAse A solution (20 µg/ml in 0.5 mol/l NaCl and 10 mmol/l Tris, pH 8.0) for 30 min at 37°C. Next the slides were rinsed in 2x SSC, 10 mmol/l beta-mercaptoethanol and 1 mmol/l EDTA and incubated in 0.1x SSC, 10 mmol/l beta-mercaptoethanol and 1 mmol/l EDTA for 2 h at 55°C. After a final wash in 0.5x SSC, sections were dehydrated, dried overnight at room temperature, dipped in Hypercoat emulsion (Amersham International Plc., UK) and exposed at 4°C for 10 weeks. Sections were developed in Kodak D19, counterstained in Carazzi's haematoxylin and 1% orcein and mounted in DPX.

2.7 Cell proliferation and tissue viability
For proliferation studies, segments of distended or non-distended vein were incubated for 24 h with culture medium supplemented with [3H]thymidine (1 µCi/ml, specific activity 25 Ci/mmol). DNA and total thymidine incorporation were determined as previously described [29]. Proliferating cells in histological sections were identified by immunostaining for proliferating cell nuclear antigen (PCNA). Briefly, sections were dewaxed and rehydrated, endogenous peroxidase was removed and sections microwaved in citrate buffer for 2x5 min sessions (700 W) in order to achieve antigen retrieval. Sections were then blocked with 1:10 dilution of goat serum in TBS followed by incubation with 1:125 dilution of PC10 antibody (Dako) in 5% BSA/TBS at 4°C overnight. This was followed by incubation with goat anti-mouse IgG in 5% BSA/TBS, TBS washes then incubation with mouse monoclonal PAP followed by DAB. Sections were counterstained with Carazzi's haematoxylin and mounted with DPX. Tissue viability before and after culture was determined by measurement of adenine nucleotides by HPLC analysis as previously described [29].

2.8 Detection of apoptosis using terminal uridine nick end-labeling (TUNEL)
The TUNEL assay was used to detect DNA fragmentation in situ. This method uses terminal deoxy-nucleotidyl transferase (TdT)-mediated nick end-labeling of DNA fragments using a commercially available apoptosis detection kit (ApoTag, Appligene Oncor), which was used with minor modifications of the manufacturer's protocol. In brief, paraffin embedded saphenous vein sections from either control or distended and non-distended segments at 24 h (3–4 µm) were dewaxed, rehydrated in graded ethanols, treated with 20 µg/ml proteinase K according to manufacturer's instructions (Promega) for 30 min at room temperature and endogenous peroxidases inactivated using 3% (v/v) hydrogen peroxide for 10 min. Subsequent labeling of DNA fragments with digoxigenin conjugated nucleotides (dUTP) and TdT used at concentrations recommended by the manufacturer was performed at 37°C, for 60 min, in a humidified chamber. After incubation for 30 min with an anti-digoxigenin peroxidase conjugate, the signal was detected using the chromogen diaminobenzidine (DAB) (Sigma) for 90 s and the slides counterstained with 0.5% methyl green for 10 min. Biochemical controls were performed with positive control slides treated with DNAse-1 (Pharmacia Biotech) and staining in the absence of TdT enzyme as a negative control.

2.9 Quantification of TUNEL and PCNA staining
TUNEL positive cells appeared brown against a methyl green background. A positive TUNEL reaction associated with dense, brown nuclear staining and cellular shrinkage was considered to be apoptosis. The number of apoptotic or proliferative cells in each high power field (x40 objective) were counted manually and represented as a percentage of the total cells in that field (i.e. TUNEL/PCNA labeling index=TUNEL/PCNA positive cells/Total number of cells per high power fieldx100). Five high power fields were randomly selected and counted from the intima (defined as the area within the internal elastic laminae), the inner longitudinal layer of the media, the outer circular layer of the media, and the adventitia (the area outside the external elastic lamina). A random selection of slides were counted by two further independent observers (CMH and SEF) in order to determine inter-observer variation.

2.10 Measurement of the thickness of the intimal and inner medial layers of the vessel wall
The thickness of the intima and the inner longitudinal layer of the media were measured using H&E-stained sections of control vein. Measurements were made with an image analysis programme (Seescan, Cambridge, UK). The thickness of each layer was calculated by averaging the thickness at ten random points around the vessel wall. The percentage apoptosis and proliferation in the outer circular layer of the corresponding distended vein was correlated with the thickness of (i) the intima, (ii) the inner longitudinal layer of the media, and (iii) the intima plus inner media using a Spearman's rank correlation.

2.11 Fos/TUNEL double staining
Fos immunostaining was performed as previously described except that the tertiary layer was replaced with alkaline phosphatase/anti-alkaline phosphatase (Dako) followed by new fuchsin substrate system (Dako). The TUNEL procedure was then carried out as described above. Counterstain was omitted and sections were mounted with aquamount (Gurr).

2.12 Electrophoretic detection of internucleosomal DNA fragmentation
Control, distended and non-distended segments of saphenous vein were snap frozen in liquid nitrogen for analysis of apoptosis by DNA fragmentation. Specimens were homogenized under liquid nitrogen and suspended in 2x NTE (200 mM NaCl, 20 mM Tris, 2 mM EDTA, pH 8.0) and 1% sodium dodecyl sulphate followed by overnight proteolysis at 37°C using 2 mg/ml proteinase K (Promega). The lysate was extracted twice with phenol/chloroform, once with chloroform alone. Samples were treated with 10 µl/ml RNAse one (Promega) and the DNA precipitated in isopropanol at 4°C overnight. The DNA was pelleted by centrifugation at room temperature, resuspended in 15 µl of distilled water with 3 µl of loading dye (0.25% orange G, 40% sucrose) and subjected to electrophoresis through a 2% agarose gel containing ethidium bromide at 80 V for 1 h. The gel was visualized under UV transillumination and photographed using a polaroid camera system.

2.13 Transmission electron microscopy detection of apoptosis
First, 3-mm segments of saphenous vein were immersion-fixed in 2% glutaraldehyde (final concentration) in 0.04 mol/l cocodylate buffer with 5% (w/v) sucrose and 0.05% (w/v) calcium chloride, post-fixed in 1.3% (w/v) osmium tetroxide, dehydrated in graded ethanols, infiltrated and embedded in LR white resin and ultra-thin sections cut. Sections were then contrasted with 2% uranyl acetate in 50% (v/v) ethanol for 30 min at room temperature and Reynolds’ lead citrate for 10 min. Sections were examined by a Philips 400 transmission electron microscope at 60 kV.

2.14 Statistical analysis of data
All descriptive statistics are expressed as mean(±S.E.M.). A paired sample Student's t-test was used to determine any significant differences in c-fos mRNA densitometry data following correction for loading with reference to 7B6 and cell proliferation rates between distended and non-distended vein. The Kruskal—Wallis one-way ANOVA followed by the Mann—Whitney U–Wilcoxon rank test was used to test for differences in labeling indices for apoptotic and PCNA positive cells between control, distended and non-distended vein. A Spearman's rank test was used to identify any correlation between vessel wall thickness and percentage apoptosis or proliferation. The Bonferroni correction was applied to adjust for performing multiple comparisons. Values of P<0.05 were considered significant. All statistical tests were performed using minitab 9.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Histology of human saphenous vein
Transverse histological sections of human saphenous vein revealed an intimal, medial and adventitial layer (data not shown). In freshly isolated vein the intima was sometimes composed of a single layer of endothelial cells lining the vessel lumen, but more commonly an intimal layer composed of circularly arranged VSMC was evident. The medial layer contained an inner layer composed of longitudinally orientated SMC interspersed with thick collagen fibres, and an outer layer of circularly arranged SMC and collagen fibres. The adventitia was composed of collagen and also bundles of longitudinally arranged SMC.

3.2 Endothelial coverage
Scanning electron microscopy of non-distended saphenous vein showed a virtually intact endothelial layer (Fig. 1a). This appearance was maintained after 24 h in serum-free culture (not shown). Distended saphenous vein showed areas of endothelial denudation and partially detached cells (Fig. 1b). This appearance was maintained following 24 h in serum-free culture (not shown).


Figure 1
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  		Fig. 1 Scanning electron microscopy (SEM) of non-distended saphenous vein showing a virtually intact endothelial layer (a). Immediately following pressure distention, SV shows areas of endothelial denudation (b). (Scale bar represents 10 µm).

 
3.3 Northern analysis
Northern analysis indicated the absence of c-fos mRNA in the control (C) saphenous vein snap frozen in liquid nitrogen immediately after harvesting. Synthesis of c-fos mRNA had occurred by 1 h in distended and non-distended vein maintained in HEPES-buffered RPMI at 0 mmHg pressure and 37°C (Fig. 2a). Cumulative densitometry data corrected for RNA loading, showed a peak of expression of c-fos at 1 h in both distended and non-distended veins. The expression of c-fos was significantly greater in the distended vein 1 h after harvesting compared to non-distended vein (Student's paired sample t-test, n=6, P<0.05) (Fig. 2b).


Figure 2
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  		Fig. 2 (a) Northern analysis showing the absence of c-fos in control saphenous vein snap-frozen in liquid nitrogen immediately after harvesting (C). By 1 h, c-fos mRNA is detectable in both non-distended (ND) and distended (D) vein. (b) Cumulative densitometry data shows a peak of expression of c-fos at 1 h in distended (white bars) and non-distended vein (black bars). At 1 h following harvesting there is a significant difference between distended and non-distended vein (* P<0.05). Error bars represent S.E.M. values.

 
3.4 Immunohistochemistry for c-Fos protein
Low levels of c-Fos protein were seen in control vein (Fig. 3a). c-Fos protein was observed in both non-distended and distended segments of vein. After 24 h, c-Fos was less abundant in non-distended vein compared to distended vein (Fig. 3b,c). The protein was mainly present in the circular media (CM), but staining to a lesser degree was also seen in the longitudinal media (LM), intima (I) and adventitia (not shown). Staining for c-Fos was mainly cytoplasmic with some positive staining nuclei (Fig. 3d). Immunostaining for c-Fos was performed with two different antibodies (see Methods). Similar results were obtained with both antibodies.


Figure 3
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  		Fig. 3 Immunohistochemical analysis of c-Fos protein expression in human saphenous vein. Micrographs show sections of control vein (a, magnification x64) snap-frozen in liquid nitrogen immediately after harvesting, where basal levels of c-Fos are visible (brown). c-Fos appears to be less abundant in non-distended vein (b, magnification x64) compared to distended vein (c, magnification x64, and d, higher power x128) 24 h after harvesting. c-Fos protein is mainly localized to the circular layer of the media (CM), but some staining is also seen in the intima (I), longitudinal layer of the media (LM) and the adventitia (not shown). (e, magnification x64) Shows a negative control with no primary antibody added.

 
3.5 In situ hybridization for c-fos mRNA
Very few cells positive for c-fos mRNA were seen in control vein snap frozen in liquid nitrogen (Fig. 4a). No positive cells were seen following hybridization with the sense probe (Fig. 4b). Maximum c-fos expression was seen at 1 h in both distended (Fig. 4c) and non-distended vein (Fig. 4d). Positive cells were located mainly in both the longitudinal and circular layers of the tunica media and around vasa vasorum within the vessel wall. c-fos expression was reduced slightly at 3 h in both distended (Fig. 4e) and non-distended (Fig. 4f) and reduced greatly at 7 h (data not shown). Less c-fos mRNA expression was detected in the non-distended vein than in the distended vein after 3 and 7 h.


Figure 4
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  		Fig. 4 Localization of c-fos mRNA in human saphenous vein by in situ hybridization histochemistry. Very few positive cells can be seen in control vein snap-frozen in liquid nitrogen (a). (b) Shows a negative control hybridized with a sense probe to c-fos. Maximum c-fos mRNA expression is present at 1 h in both distended (c) and non-distended (d) vein with similar distributions. Positive cells are located mainly in the media and around vasa vasorum in the adventitia. c-fos expression is reduced at 3 h and at this time point it is more widely distributed in distended vein (e) compared to non-distended vein (f). c-fos expression is reduced again at 7 h (not shown). I, intima; CM, circular intima; LM, longitudinal media. Magnification x64.

 
3.6 Cell proliferation and tissue viability
[3H]Thymidine was incorporated into both distended and non-distended veins cultured for 24 h. This incorporation was significantly higher in the non-distended than in the distended veins: mean (S.E.M.): 1279 (218) vs. 863 (155) dpm/µg DNA (Student's paired sample t-test, n=5, P<0.05). PCNA immunostaining showed mitotic cells in all vein segments with no significant differences in labelling indices between the control, distended and non-distended (Table 1). Adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratios were determined to assess cell viability of segments of saphenous vein. The ATP/ADP ratios remained high during culture in both distended and non-distended vein segments indicating that the tissue remained viable during the 24-h culture period in both distended and non-distended vein segments (data not shown). The DNA concentration of distended and non-distended vein segments declined slightly following 24 h in culture (control=0.19 µg (0.04) DNA/mg tissue), although there was no significant difference between the distended and non-distended specimens, i.e. 0.13 (0.02) vs. 0.12 (0.01) µg DNA/mg tissue, respectively.


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  		Table 1 PCNA-labelling index for proliferating cellsa

 
3.7 Detection of apoptosis
TUNEL positive cells were identified by dense brown staining associated with other light microscopic features of apoptosis including cell shrinkage, preserved cell membrane, rounded nucleus and occasional apoptotic bodies. Control segments of saphenous vein showed only very occasional TUNEL positive cells within the intima, media and adventitia (Fig. 5a). Following 24-h incubation in serum-free media, the labeling indices increased but still very few apoptotic cells were identified in the non-distended vein segments (Fig. 5b). Distended vein however, showed a significant rise in the apoptotic labeling index in the media compared to both control and non-distended vein, (Kruskal—Wallis one-way ANOVA and Mann—Whitney U-test, n=5, P<0.05) (Figs. 5c and 6Go). Increased levels of apoptosis were seen in the circular layer of the media in distended vein (7.32±3.18%) compared to the longitudinal layer (2.59±1.38%), however, this difference was not significant (Kruskal—Wallis one-way ANOVA). No significant differences between control, non-distended and distended vein were seen in apoptotic index in the intima or adventitia. Similar data were obtained when slides were counted by two further independent observers. Negative control slides (see Methods) showed no positive cells (Fig. 5d). When the intimal and inner medial thicknesses were compared with proliferation or apoptosis no significant correlations were observed. Double staining for Fos and TUNEL demonstrated positive staining for both, however co-localisation was not observed (data not shown).


Figure 5
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  		Fig. 5 Detection of apoptosis by TUNEL. TUNEL positive cells were identified by intense brown staining associated with other light microscopic features of apoptosis (arrowheads). Micrographs show sections of (a) control vein snap-frozen in liquid nitrogen immediately after harvesting, where occasional TUNEL positive cells are seen, (b) non-distended vein 24 h after harvesting, and (c) distended vein 24 h after harvesting. (d) Shows a control slide with no TdT enzyme added. I, intima; CM, circular media; LM, longitudinal media. Magnification x64.

 

Figure 6
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  		Fig. 6 TUNEL labeling indices (means) of apoptotic cells in control (n=5), non-distended (n=5), and distended (n=5) segments of saphenous vein. Error bars indicate standard error of the mean. Means with the same letter are significantly different from each other (Mann—Whitney rank sum test, P<0.05, adjusted for multiple comparisons with the Bonferroni correction).

 
To confirm that TUNEL staining specifically detected apoptotic cells, vein segments were also examined by transmission electron microscopy (data not shown). Apoptotic cells with characteristic morphology, including cell shrinkage, membrane blebbing, chromatin condensation and apoptotic bodies, were seen in distended, and to a lesser extent in non-distended vein, 24 h after vein harvesting. Apoptosis was detected in both endothelial cells and VSMCs. In addition, DNA fragmentation was detected by gel electrophoresis of genomic DNA isolated from distended or non-distended segments of saphenous vein. Characteristic laddering, representing DNA fragmentation was detected in the distended saphenous vein segments but was absent in control and non-distended segments of vein (Fig. 7).


Figure 7
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  		Fig. 7 Gel electrophoresis of genomic DNA showing DNA fragmentation in distended vein 24 h after harvesting (D), but not in control (C) or non-distended (ND) vein. The 100-bp ladder (Promega) indicates 500-bp size fragment (arrow).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of this study reveal that freshly isolated (control) segments of saphenous vein do not express c-fos mRNA. Surgical harvesting of vein leads to the expression of c-fos mRNA as early as 1 h after isolation. These findings are similar to a recent study by Moggio et al. [30] In the present study, c-fos expression was higher and more sustained in distended compared to non-distended vein. C-Fos protein was also investigated using immunohistochemistry and was present within the medial, and to a lesser extent, the intimal and adventitial cells of vein sections. In parallel with mRNA findings, c-Fos protein was higher in the distended compared with the non-distended and control saphenous vein.

In order to localize c-fos mRNA within the vessel wall, in situ hybridization was performed. The majority of c-fos mRNA was detected within the media. Our data is in agreement with the findings of Okazaki et al. [31] who examined c-fos expression in rat aorta and detected low basal levels of c-fos with increased expression after activation with phenylephrine primarily in the medial smooth muscle layer rather than in the adventitial fibroblasts. Previous studies have demonstrated activation of c-fos and c-jun following arterial injury in a rabbit and a rat model of balloon de-endothelialisation [23,24]. In these models, smooth muscle cell proliferation, migration and intimal thickening subsequently occur in a similar process to the late failure of saphenous vein grafts. Antisense RNA to c-fos has been shown to prevent cell entry into s phase and hence inhibit cellular proliferation [32]. However, in contrast to this, targeted disruption of the c-fos gene demonstrates that a deficiency in c-fos does not significantly affect fibroblast growth and normal AP-1 DNA binding activity is shown in these cells [12].

In our study, the upregulation of c-fos in distended human saphenous vein correlates with an increase in apoptosis and a decrease in [3H]thymidine uptake. We failed to detect a change in PCNA positive staining between distended and non-distended saphenous vein. This is probably due to the problems associated with using this technique, i.e. that because of its long half-life, PCNA immunostaining is known to overestimate the number of proliferating cells [33].

Previous work performed on cardiac myocytes has shown that mechanical stretch may provide the stimulus for apoptosis to occur [34], and recent work in our own laboratory has demonstrated the induction of apoptosis in porcine coronary arteries following balloon injury which preceded cell proliferation [35]. A pronounced loss of SMC has previously been reported in the circular layer of the media of human saphenous vein grafts, with the inner longitudinal layer being less affected 24 h following grafting [36]. Ischaemia and interruption of the vasa vasorum have been suggested as the causes but it is likely that when stretch, or distension, is applied the circular layer of smooth muscle cells in the media are more severely damaged. The data presented here do not show a statistical relationship between the intimal and inner medial thicknesses and the proliferation and apoptotic indices. This may be for a number of reasons. First, the time of incubation may be insufficient for morphological consequences to develop. The second reason is that there is a complex relationship between proliferation and apoptosis that is under tight control to ensure that a correct response occurs. The third possibility is that there is no relationship between proliferation or apoptosis and the morphological response, the latter arising purely as a result of changes in extracellular matrix.

A number of studies have demonstrated an association between c-fos and apoptosis [22,37,38], however, studies using mutant mice lacking functional copies of the genes for c-fos and c-jun have shown that c-fos is not essential for apoptosis [39,40]. In the present study, we did not identify co-localisation of TUNEL positive cells and c-Fos protein. It is possible that the timing of apoptosis and c-Fos protein may not be concordant at the time-point investigated (i.e. 24 h). Also, a recent study has shown c-Fos protein to be degraded when mouse lymphoma cells were induced to undergo apoptosis [41] and this could also explain the lack of co-localisation.

The results of this study show that the oncogene c-fos is induced following removal of human saphenous vein prior to coronary artery bypass surgery and that the induction of this gene is temporally associated with apoptosis that is known to occur in saphenous vein graft failure. This finding provides a rationale for manipulating the expression of these genes to prevent vein graft failure. Candidate reagents include angiopeptin, a synthetic analogue of somatostatin, which has been shown to inhibit the induction of c-fos and c-jun in rabbit aorta after balloon denudation as well as inhibiting intimal proliferation in various animal models and in clinical trials [42,43]. Other studies using antisense oligonucleotides have demonstrated that this approach is applicable in the treatment of saphenous vein grafts [44]. Furthermore, angiotensin II type-1 receptor blockade has been shown to inhibit the expression of c-fos and c-jun [45]. The findings of this study indicate that c-fos, being linked to proliferation and apoptosis, is a legitimate target for manipulation to prevent late saphenous vein graft failure.

Time for primary review 27 days.


   Acknowledgements
 
The expert assistance of Ian Palmer, Electron Microscopy Unit, Department of Pathology, University of Sheffield is gratefully acknowledged, as is that of theatre staff at the Northern General Hospital, Sheffield. This research was funded by grants from the Northern General Hospital Trust Research Committee and the Special Trustees of the Former United Hospitals of Sheffield. JA is recipient of a University of Sheffield, Faculty of Medicine Scholarship.


   Notes
 
1 These authors have contributed equally to the manuscript. Back


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

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