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Ann Thorac Surg 2006;82:1458-1464
© 2006 The Society of Thoracic Surgeons
a Crafoord Laboratory, Karolinska Hospital, Stockholm, Sweden
b Department of Thoracic Surgery, Karolinska Hospital, Stockholm, Sweden
c Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
d Department of Surgery, Ulleval University Hospital, Oslo, Norway
Accepted for publication May 11, 2006.
* Address correspondence to Dr Valen, Institute of Basic Medical Science, Department of Physiology, University of Oslo, Postbox 1103 Blindern, 0317 Oslo, Norway (Email: guro.valen{at}medisin.uio.no).
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Abstract |
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METHODS: Biopsy samples of saphenous veins were taken from 9 patients undergoing elective CABG at the start of vein harvesting (open technique) and after the last proximal anastomosis was sutured. Messenger RNA was extracted and amplified with semiquantitative reverse transcription polymerase chain reaction. Gene expression of proinflammatory cytokines (tumor necrosis factor-
, interleukin-1ß), leukocyte adhesion molecules (E-selectin, intercellular adhesion molecule-1), and vasoactive substances (endothelin-1, inducible and endothelial nitric oxide synthase) was investigated. Translocation of nuclear factor-
B (NFB) was evaluated with electrophoretic mobility shift assay. Immunostaining for von Willebrand factor was performed to evaluate loss of endothelium, and in vitro vein reactivity to phenylephrine and endothelin-1 was studied.
RESULTS: Gene expression of cytokines and leukocyte adhesion molecules increased after graft harvesting and storage, whereas vasoactive substances did not change. Nuclear translocation of NFB occurred after surgical handling, concurrent with partial loss of endothelium and impaired contractile function.
CONCLUSIONS: Standard surgical handling of vein grafts induces NFB-driven inflammation in the vessel wall and impairs vascular function. This may potentially contribute to both early and late graft occlusion.
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Introduction |
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The causes of venous graft occlusion—intimal hyperplasia and atherosclerosis—are not fully elucidated. The transition from a venous to an arterial position, with shear stress, pulsatile flow, and high pressures, may contribute [3, 4]. Some evidence indicates that the process of harvesting and preparing the vein graft may contribute to graft injury and possibly both early and late occlusion [5, 6]. After surgical handling, there is functional and morphologic injury to the endothelium as well as endothelial denudation [7, 8]. The graft preservation fluid, storage temperature, surgical technique, and the distension pressure to which the vein graft is exposed during surgery seems to be important for the inflicted injury [7–12].
The cellular and molecular changes induced in the vessel wall during surgery are not well characterized. Surgical handling induces protein expression of intercellular adhesion molecules [13, 14]. Expression of the early immediate genes encoding for c-fos and c-myc are induced, as is vein wall apoptosis [15, 16]. Injury to the endothelium and the vessel wall may induce a prothrombotic state that causes early graft occlusion. Further insight into the molecular events occurring during vein graft harvesting and handling may provide tools to modulate graft injury and increase patency.
Nuclear factor- B (NFB) may be regarded as the orchestral leader of the inflammatory response [17]. It is activated by more than 150 stimuli and regulates transcription of more than 150 genes. Many of the genes NFB regulates are key factors driving leukocyte attraction, adherence, and migration; proinflammatory cytokines such as tumor necrosis factor- (TNF) and interleukin-1ß (IL-1ß), leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin (CD62E), as well as regulators of vascular tone such as inducible nitric oxide synthase (NOS2) and endothelin-1 (ET-1) [17]. It is currently not known if an inflammatory response regulated by NFB is involved in vascular injury after surgical handling.
We hypothesized that standard vein graft harvesting and handling caused an inflammatory response in the vessel wall with translocation of NFB and expression of some proinflammatory genes it regulates. Loss of endothelium, gene expression of endothelial nitric oxide synthase (NOS3), and vein graft function in vitro were evaluated.
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Material and Methods |
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Anesthesia and Surgery
Because vein grafts were the objects of the present study, the surgical procedures on the heart are not given in detail. All patients were anesthetized according to department's clinical routine. After premedication with morphine, anesthesia was induced with fentanyl, midazolam, and propofol, and muscular relaxation was achieved with pancuronium or atracurium. Volume-controlled ventilation with 40% to 50% oxygen in air was performed. Anesthesia was maintained with intermittent fentanyl and isoflurane, and continuous propofol was used as a supplement when needed.
The operations were done through standard median sternotomy with (7/9) or without (2/9) cardiopulmonary bypass (CPB). For CPB, heparin (300 IU/kg) was administered to obtain an activated clotting time exceeding 480 seconds. Antegrade cold blood cardioplegia was used. The patients received 3.2 ± 0.2 anastomoses. The time on CPB was 99 ± 19 minutes, and the duration of aortic cross-clamping was 70 ± 17 minutes. Patients operated on off pump initially received heparin 150 IU/kg, and their activated clotting times were kept above 300 seconds while the anastomoses were performed. All proximal anastomoses were performed by the use of a side-biting clamp in both on-pump and off-pump surgery.
Vein Sampling
All veins were harvested with an open technique by one junior surgeon. The first biopsy specimen was sampled from the distal end of the saphenous vein immediately after the skin incision, before any further dissection or handling. After harvesting of the whole graft material, it was prepared, checked for leakage, and stored in Ringer's acetate without any additives at room temperature until implantation. Leakage was checked by high pressure flushing (100 to 150 mm Hg) with a 10 mL syringe filled with Ringer's acetate. Leaks were repaired with 8-0 Prolene (Ethicon, Somerville, NJ) suture.
The intact vein graft was divided into individual grafts after each distal anastomosis to the heart was complete. The second biopsy specimen was a leftover vein after completion of the last proximal anastomosis and thus was collected from a more proximal segment of the vein. The samples were divided into one RNAse-free tube, immediately transferred to dry ice, and frozen at –80°C until processed. Another sample was transferred to a tube with 4% formaldehyde for immunohistochemistry, and the third sample was kept in Krebs Henseleit solution and immediately transferred to the laboratory for in vitro reactivity studies. The time from the first to the second sample was 126 ± 21 minutes. The vein was harvested from all patients before the start of CPB.
Preparation of Nuclear Protein Extracts
Frozen vein grafts were homogenized in a microdismembrator (Braun Biotech International, Melsungen, Germany). The samples were effectively disrupted before adding lysis buffer and protease inhibitor mix and homogenized. After 15 minutes' incubation on ice, nuclei were collected by centrifugation for 1 minute at 8000g and the supernatant discarded. The pellet containing the nuclei was washed with 20 mmol/L KCl buffer and centrifuged for 1 minute at 8000g. The isolated nuclei were resuspended in 20 mmol/L KCl buffer and 0.6 mol/L KCl was added. The nuclei were kept at 40°C for 30 minutes. After centrifugation for 15 minutes at 8000g, the supernatant containing nuclear proteins was collected. The protein concentration was determined by using a bicinchonic acid reagent (Pierce, Rockford, IL) with bovine serum albumin as a standard.
Electrophoretic Mobility Shift Assay
Nuclear extracts (16 µg protein/lane) were preincubated for 10 minutes in binding buffer (20 mmol/L Hepes, pH 7.9, 5% glycerol, 5 mmol/L MgCl2, 0.5 mmol/L ethylene diamine pentaacetic acid, and 1 mmol/L dithiothreitol) on ice, followed by 30 minutes' incubation at room temperature with 50,000 cpm of 32P-labelled probe containing the NFB binding site 5' AGT TGA GGG GAC TTT CCC AGG C (Promega Biosciences, Inc, San Luis Obispo, CA). DNA-protein complexes were electrophoresed on a 4% polyacrylamide gel. For supershift analysis, rabbit polyclonal anti-p50 and anti-p65 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with the binding buffer for 15 minutes before adding the radio-labelled probe. For competition analysis, an unlabelled probe in 50-fold or 100-fold excess was added before the radio-labelled probe. Non-sense competition was performed with preincubation with a cold AP-1 probe (Promega). Band densities were calculated from autoradiographs by using TINA 2.0 computer software (Raytest, Straubenhardt, Germany).
Messenger RNA Extraction and Complementary DNA Synthesis
Frozen tissue was homogenized in a microdismembrator and mRNA extracted using a Dynabeads messenger RNA (mRNA) direct kit (Dynal A.S., Oslo, Norway), with the protocol supplied by the manufacturers. Single-stranded complementary DNA (cDNA) synthesis was performed by Superscript II (Life Technologies, Paisley, UK), according to the manufacturer, using random hexamers (Life Technologies) as primers in the presence of RNasin (Promega, Madison, WI).
Semiquantitative Polymerase Chain Reaction
The principle of this reaction as well as the sequence of primers is described in detail elsewhere [18]. Briefly, gene expressions of all mediators and in both biopsy samples were performed on the same mastermix in one patient to avoid differences due to pipetting of small volumes. Each polymerase chain reaction (PCR) was run in a volume of 25 µL. A mastermix was prepared consisting of dNTP (0.25 mmol/L), MgCl2 (1.5 mmol/L or 3.0 mmol/L for ICAM-1, ET-1 and NOS2), PCR buffer, 0.02 U Taq polymerase (all Life Technologies), and 10 µCi 33P-dATP/sample (Amersham Pharmacia Biotech, UK). After the mastermix was divided, cDNA was added, samples were aliquoted in separate PCR tubes, and primers were added at a final concentration of 0.2 µmol/L. Histone H3, which is expressed at the same level independent of cell cycle, was selected as a housekeeping gene and amplified in all samples.
Amplification was started in a hot block at 94°C for 2.5 minutes, 60°C for 30 seconds, and 72°C for 45 seconds, followed by cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds for H3, TNF, IL-1ß, CD62E, ICAM-1, and NOS3. For ET-1 and NOS2, an annealing temperature of 65°C was used. The linear phase of the PCR reaction was determined for each gene in four individual samples, and the number of cycles selected was kept constant throughout the study [18]. A control PCR of mastermix without cDNA and with the H3 primer was routinely done in all samples, whereas control reactions on RNA were performed randomly to identify possible contamination.
A radio-labelled DNA ladder was synthesized using the Gibco 100 base pair DNA ladder and T4 DNA polymerase kit according to the manufacturer's instructions (Life Technologies), with 33P-dATP as the incorporated marker. The PCR products were separated by electrophoresis on a 5% polyacrylamide gel and analyzed in a phosphoimager (Bioimaging Analyzer System BAS 1000, Fuji, Tokyo, Japan). The ratio between the optical density of test gene and the H3 band was calculated to evaluate relative changes in expression of the test gene using the TINA 2.0 computer software.
Immunohistochemistry
Vein grafts from 6 patients were used for immunohistochemical analysis. The vein grafts were fixed in 4% formaldehyde overnight and stored in phosphate-buffered saline (PBS) with 10% sucrose until staining. The tissue was dehydrated and embedded in paraffin. Sections 5-µm thick were deparaffinized and hydrated with xylene and ethanol. After high temperature antigen retrieval and treatment with blocking solution (5% goat serum) for 30 minutes, the sections were incubated with a mouse polyclonal antihuman von Willebrand factor (vWF) Ab, F8/86 (1:400, DAKO, Glostrup, Denmark) overnight at 4°C, and thereafter, secondary antibody, biotinylated horse anti-mouse immunoglobulin (Vector Laboratories, Burlingame, CA) was added. An avidin-biotin detection system (Vectastain Elite ABC Kit, Vector Laboratories) and diaminobenzidine tetrahydrochloride (DAB Substrate Kit, Vector Laboratories) were used for visualization. Hematoxylin was used for counterstaining. The length of vWF-positive endothelium in the lumen was measured and calculated as percentage of total lumen length by using Photoshop (Adobe Systems, Inc, San Jose, CA). All measurements were made by the same investigator in a blinded fashion.
Vessel Reactivity Study
Vein graft samples were placed in Krebs Henseleit solution at room temperature. The Krebs Henseleit solution consisted of (in mmol/L): NaCl, 118; KCl, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4(7H2O), 1.2; NaHCO3, 25.2; and glucose, 11.1. The graft was rapidly transferred from the operating room to the laboratory, cut into 2-mm-long rings, and mounted onto two thin stainless steel holders, one of which was connected to a force displacement transducer (Grass model FT.03, Grass Instrument Co, Quincy, MA). A passive tension of 1g was used, which was determined to be the optimal resting tension for obtaining the maximal active tension induced by 127 mM K+ solution. The mounted rings were kept at 37°C in 2 mL organ baths containing Krebs Henseleit solution, and continuously bubbled with a gas mixture of 95% oxygen and 5% carbon dioxide to maintain a pH of 7.4. The isometric tension was recorded on a polygraph (Grass model 7B).
After a 60-minute equilibration period, the contractile function of the vessel was tested twice with 127 mM K+ solution, and the mean was taken as a maximum contraction. After washout, contractile responses to the selective 1-adrenoceptor agonist phenylephrine (10–8.5 to 10–5.0 mol/L) (Sigma Chemical, St. Louis, MO) and endothelin-1 (ET-1, 10–11.0 to 10–6.0 mol/L) (Alexis Corporation, Läufelfingen, Switzerland) were determined. In pilot studies, endothelium-dependent and endothelium-independent relaxation of veins precontracted with 1 µmol/L phenylephrine was attempted. However, all relaxation was abolished after surgical preparation, and the attempts were discontinued.
Statistics
Gene expression data at the start and end of surgical handling are presented as box plots with median values, 25 and 75 percentiles, and range. Wilcoxon signed-ranks test was used to evaluate differences between samples. The vessel reactivity data are presented as mean ± SD. The vessel reactivity data, optical density data, and immunohistochemical measurements were analyzed by a one-way analysis of variance combined with the least significant difference test for post hoc comparisons. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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BB, and separated by electrophoresis. Two different electrophoretic mobility shift assays with samples from 6 patients altogether are shown in Figure 1. In the vein samples collected after surgical handling, the optical density of the bands increased in most of the patients, indicating activation of NFB (Fig 1). The identity of the proteins bound to the probe were determined by supershift analysis and cold probe competition (Fig 1). Antibodies specific for the p50 and p65 subunits of the NFB heterodimer caused retardation of the mobility of the DNA probe, whereas the band disappeared when a cold probe was used, further identifying the band as NFB. Further support of band identity was that preincubation with cold AP-1 probe did not influence mobility (Fig 1). When band densities of all patients were measured and calculated, an increased NFB activation after surgical preparation was apparent (from median arbitrary band density of 620 units to a median density of 1100, p = 0.028).
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, IL-1ß, CD62E, and ICAM-1 increased after the surgical handling and storage (p < 0.05; Fig 2B). Gene expression of the vasoactive factors NOS2, NOS3, and ET-1 did not change after surgical preparation (Fig 2B).
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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B-driven inflammatory response was induced in the saphenous vein as a result of standard surgical preparation of the graft before anastomosis to the coronary arteries. Morphologically, the vein grafts were dilated and flaccid. This was accompanied by partial loss of endothelium and loss of endothelial function evaluated by in vitro reactivity. Functionally, the implanted veins grafts had no reactivity. This study of the effects of vein graft handling combining analysis of vascular morphology, function, and molecular biology in the vein samples indicates that the long-term low patency of vein grafts may be a result of a process that has already started in the operating theater with the act of harvesting and handling the vein.
Gene expressions of proinflammatory cytokines and leukocyte adhesion molecules were upregulated by surgical handling. The expression of these genes could be explained by activation of the transcription factor NFB, because they contain motifs for NFB in their promoter regions [17, 19]. Other transcription factors not measured in the present study may have interacted in the gene regulation. The latter is likely, as the genes were not induced to the same extent. For instance, NOS2, which contains a motif for NFB but also for other factors such as hypoxia-inducible factor 1 [17], was not upregulated in the present study. That is in accordance with a previous study on NOS2 protein expression in human vein grafts, which did not increase by surgical handling [13].
We do not know the exact stimuli leading to nuclear translocation of NFB in the present study, but can speculate that a shear-stress response may have been involved. Shear-stress response elements are found in promoter regions of, for instance, ICAM-1 [13], and NFB activation induced by shear stress has been described in experimental studies [16, 17, 19].
The induction of genes encoding for adhesion molecules and cytokines is characteristic of inflammatory responses. Adhesion molecule expression promotes the recruitment of cytokine-producing leukocytes, and both TNF and IL-1ß induce adhesion molecule expression via NFB activation in a positive feedback loop [17, 19]. Transfection of vein grafts with a NFB decoy reducing the inflammatory response during surgery reduced neointima formation in a canine model [20].
The success of CABG is based to a large extent upon the use of saphenous vein grafts. The vascular endothelium plays an important role for vascular integrity. Endothelial cells serve as an antithrombogenic lining of the vessel wall, and produce an abundance of modulatory substances in response to mechanical and hormonal signals, among them nitric oxide, prostacyclin, endothelium-derived hyperpolarizing factor, thromboxane A2, prostaglandin H2, angiotensin II, and ET-1. Endothelium-derived mediators regulate vascular tone, proliferation of vascular smooth muscle cells, and leukocyte adhesion as well as platelet adhesion and aggregation, all of which are important factors for preserving vein graft patency [3–6].
Vein grafts are likely to continue to have an important role for CABG in the immediate future. Thus, optimizing the procedures for handling and preservation of graft material may contribute to preserve the endothelium, limit the inflammatory reaction, and possibly increase graft patency. We found loss of endothelium evident as reduced vWF-stained areas after surgical handling. This is in accordance with previous reports on endothelial integrity after CABG procedures [7–12, 20].
Vein function was severely impaired after surgical preparation; both relaxation as well as contraction virtually disappeared. This finding confirms those observed by Fabricius and colleagues [21] and Engstrom and colleagues [22]. When the veins were harvested with minimally invasive techniques, in vitro reactivity was preserved [22, 23]. Results of electron microscopic studies have suggested that a more gentle technique of vein harvesting [7] or improved storage of the vein [8, 11] protects the endothelium.
A limitation to this study is that the time frame of observation was short, and a larger inflammatory induction is likely to occur in vivo outside of the time frame where we may take biopsies. Furthermore, we cannot address the question of which stimulus during graft preparation was of highest importance for the responses. Shear stress, Ringer's acetate, room temperature, and mechanical handling are all possible contributing factors.
The inflammation of the vein graft wall may contribute to both early and late graft occlusion, but a further limitation of this study is that the patient population is too small to do an in vivo follow up of graft patency in relation to the inflammatory response. The major changes and loss of function after standard surgical handling is a surprising observation. The utmost attention must probably be paid to the techniques of dissection, surgical handling, and storage of vein grafts during CABG. Probably relatively simple technical changes may have considerable impact on graft patency, which may be further improved by modulation of the molecular changes induced during vein preparation.
In conclusion, a NFB-driven inflammatory response is induced in the vein wall after standard surgical preparation. Nuclear translocation of NFB and increased expression of the genes it regulates concurred with loss of endothelial integrity and function.
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Acknowledgments |
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