Eur J Cardiothorac Surg 2004;26:995-1001
© 2004 Elsevier Science NL
Non-viral in vivo thrombomodulin gene transfer prevents early loss of thromboresistance of grafted veins
Noriyuki Tabuchia,
Masayoshi Shichiric,*,
Aya Shibamiyab,
Takatoshi Koyamab,
Fumie Nakazawab,
Jihwa Chungb,
Shinsaku Hirosawab,
Makoto Sunamoria
a Department of Cardio-Thoracic Surgery, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
b Department of Allied Health Sciences, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
c Tokyo Medical and Dental University Medical Hospital, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Received 18 February 2004;
received in revised form 2 July 2004;
accepted 8 July 2004.
* Corresponding author. Tel.: +81-3-5803-4571; fax: +81-3-5803-0254. (E-mail: mshichiri.cme{at}tmd.ac.jp).
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Abstract
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Objective: Immediate loss of thrombomodulin activity in the endothelium of vein grafts has been demonstrated during 90min exposure to arterial circulation; this loss of activity is ascribed as an important cause of early thrombosis. Conventional ex vivo gene transfection after vein harvest cannot cover this acute period immediately after implantation. We have established a highly efficient non-viral gene therapy protocol utilizing modified transferrin receptor-facilitated gene transfer. Using this technique, we examined whether in vivo thrombomodulin gene therapy, directed to the endothelium of rat veins 2 days prior to grafting, may prevent thromboresistance impairment of vein grafts under simulated arterial circulation. Methods: Abdomen of SD rat was opened and cationic liposome:transferrin:thrombomodulin gene complexes or the vector without DNAs were applied to the inferior vena cava of rats while blood flow was reduced by proximal and distal clamping. After 2 days, the transfected veins were harvested and thrombomodulin expression and thromboresistance properties determined before and after exposure to an artificial circuit. Results: The trial of gene transfection using variable doses of DNAs confirmed that 7.5µg of total DNAs was the most efficient quantity for thrombomodulin gene transfection to IVCs, although accompanying an increase of gene expression in other downstream organs. By transfection of the thrombomodulin gene in IVCs, the generation capacity of activated protein C in venous endothelium increased three-fold compared with veins treated with vector alone (P<0.01). Under simulated arterial circulation, perfusion of veins treated with vector alone decreased thrombomodulin activity to 36% of preperfused levels (P<0.01), whereas transfected grafts preserved the activity at normal vein endothelium levels even after perfusion. Consequently, the increase in endothelial thrombin activity induced by simulated arterial circulation was markedly attenuated in transfected veins (P<0.01), while immunohistochemistry confirmed the preservation of endothelial lining. Conclusions: Transferrin receptor-facilitated in vivo gene transfer to the inferior vena cava resulted in sufficient thrombomodulin gene expression immediately after graft implantation and subsequent maintenance of thromboresistance even after exposure to arterial pressure. Although further studies are needed, the present results suggest the possibility of gene therapy targeting acute phases of vein graft disease.
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1. Introduction
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Early occlusion by thrombosis occurs more frequently in vein grafts than in arterial grafts [1]. This has been partly attributed to the loss of endothelial thromboresistance associated with exposure to arterial circulation [2–4]. Thrombomodulin (TM) plays an important role in maintaining endothelial thromboresistance by inactivating thrombin and activating the protein C system. TM activity in vein grafts have been shown to decrease dramatically upon exposure to 90min of arterial shear stress [3]. Vein grafts have been shown to be good targets for gene therapy, because the explanted veins are available for ex vivo gene transfer before grafting. Restoration of TM expression by adenovirus-mediated TM gene transfer upon graft implantation has succeeded in enhancing the graft capacity to activate protein C from day 3 [4]. Theoretically, however, it would be ideal if the TM gene could be transfected in vivo into the saphenous vein prior to the grafting operation to achieve maximal TM expression immediately after graft implantation, because loss of thromboresistance takes place even during 90min of arterial perfusion [3]. Such in vivo gene therapy under flow conditions should be highly efficient for attaining sufficient TM expression compared to that under the completely static conditions that are usually used [4,5]. At the same time, it would be free from the serious adverse reactions that have recently been reported for adenoviral gene therapy [6].
Non-viral gene therapies can avoid the immune system reactions associated with viral vectors, but their low efficiency has limited the use of non-viral vectors for gene therapy [7]. The development of ligand-facilitated transfer of cationic liposome: DNA complexes has achieved some increase in in vitro gene delivery efficiency [8]. However, an even more dramatic increase in gene delivery efficacy has been obtained with our modified protocol, in which the introduction of transferrin allowed rapid and efficient uptake of the complexes by cells that were initially in direct contact, resulting in expression of the exogenous cDNA to a level producing a systemic therapeutic outcome without eliciting immune responses [9].
The present study was designed to determine whether in vivo transfection using ligand-facilitated transfer of cationic liposome:TM gene complexes into the endothelium of the inferior vena cava (IVC) prior to vein grafting operations could efficiently prevent the loss of thromboresistance in a rat vein graft model of ex vivo perfusion.
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2. Materials and methods
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2.1. Plasmids and reagents
A 2.6-kb Xho I-Bal I fragment of the human TM gene containing 5'- and 3'-untranslated and coding regions was used to construct an expression vector, pPCITMN [10], which was transcribed under the control of the cytomegalovirus enhancer and promoter (Promega, Madison, WI). Synthetic cationic liposomes, (+)-N,N bis (2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide, were purchased from Promega, and human holo-transferrin was from Sigma Chemical Co. (St. Louis, MO). Cationic liposome:pPCITMN:transferrin complexes (CLTMTC) were prepared essentially as described [11,12]. The total amounts of DNA, cationic liposome, and transferrin used for the following rat experiment were carefully determined according to the previous studies [8,13]. All other chemicals were reagent-grade products purchased from Wako Pure Chemicals (Osaka, Japan) or Sigma (St Louis, MO), unless otherwise indicated.
2.2. Animal experiments
All animals were treated under protocols approved by the Animal Care and Use Committee of Tokyo Medical and Dental University and received humane care in compliance with the European Convention on Animal Care. The experiments were carried out in accordance with the Guidelines for Animal Experimentation in Tokyo Medical and Dental University. Male Sprague–Dawley (SD) rats weighing 250–300g were anesthetized with an intraperitoneal injection of ketamine (40mg/kg). The IVC was exposed through a midline incision, the left renal vein ligated, and a small arteriotomy cannula (DLP31002, Medtronic Inc., Minneapolis, MN) introduced proximally to the IVC. While applying the temporary clamp above the take-off of the left renal vein, the IVC was gently flashed with 2ml of warm saline containing heparin (1mg/kg body weight) to obtain local hemodilution. Then, another clamp was applied distally above the bifurcation, and 1.5ml of cationic liposome:pPCITMN:transferrin complexes (7.5µg of DNAs) or liposome:transferrin complexes without DNA was slowly injected. No side branches of the IVC were interrupted except for the left renal vein, although the main blood stream was closed by temporary clamps, which allowed a limited blood flow in and out during clamping. The temporary clamps were applied for 30min to facilitate transfection. After the blood flow was restored, the peritoneal cavity was irrigated with warm saline and the wounds closed with 4-0 silk sutures. After 48h, the rats were anesthetized again and the transfected IVCs harvested after the injection of heparin (1mg/kg body weight). For Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the superior vena cava (SVC), lung, heart, kidney, and liver were removed immediately after sacrifice. Heparinized blood was also collected from the abdominal aorta to detect the expression in blood cells. Five transfected rats and 5 control rats were prepared for each of the following assays.
2.3. Ex vivo flow circuit
The excised veins were cannulated (DLP31002) and placed in an ex vivo arterial flow circuit (Senko-Ika Ltd, Tokyo, Japan) with a roller pump (Masterflex 7518-10; Cole-Parmer Inc., Vernon Hills, IL). 50% diluted and oxygenated blood was perfused for 90min with a continuous pressure of 70mmHg (flow rate, 40ml/min). The wall shear stress, as calculated by the equation for nonpulsatile flow: 4µQ/
r3 (where µ is the viscosity of the perfusate, Q is flow volume in ml/s, and r is the radius of the graft in cm), was 4dyn/cm2. The wall tension on vein graft during perfusion, as estimated from an equation: PR (where P is intraluminal pressure in dyn/cm2 and R is vessel radius in cm), was approximately 20x103dyn/cm. The blood was collected just before the harvest of the vein after heparinization (1mg/kg body weight). After 90min, the veins were removed and subjected to histological analyses or assays of activated-protein C/thrombin activities.
2.4. Western blotting
After harvest, the organ tissues were washed with phosphate-buffered saline (PBS). To prepare cell lysates, the excised veins were sonicated in lysis buffer containing 1% Triton X-100, 100µg/ml PMSF, and 0.1mol/l NaCl in 20mmol/l Tris–HCl (pH 7.5) as described previously [4]. Cellular debris was removed by centrifugation at 12,000xg for 20min. After centrifugation, the lysates were assayed for total protein content using a BCA protein assay kit (Pierce, Rockford, IL). The cell lysates were stored at –80°C until use. Ten micrograms of each sample was electrophoresed through a 10% SDS-polyacrylamide gel and transferred to a nylon membrane (Boehringer Mannheim, Germany). TM antigens in the cell lysates were detected by Western blotting, using a rabbit anti-human TM polyclonal antibody (Santa Cruz Biotechnology Inc., CA), and quantified by densitometric analysis with Scion Image software (Microsoft). The results were presented as the ratio of the density of the bands of IVCs simultaneously taken from rats without any treatment.
2.5. Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Organ tissues were washed with PBS, and total RNAs were isolated using RNA-Bee (TEL-TEST Friendswood, TX). The TM mRNAs in 0.1µg of each sample were detected by RT-PCR, using a Titan One Tube RT-PCR Kit (Roche Diagnostics, Mannheim, Germany). Amplification of human TM transcripts was performed using specific primers (forward primer: 5'-CAT GTG CGA GAC CGG CTA CCG GCT GGC GG-3', and reverse primer: 5'-AGG GGC TGG CAC TGG TAC TCG CAG TTG GC-3'). The reaction produced a 218-bp PCR product that could be confirmed by agarose gel electrophoresis. These primers were confirmed to recognize the corresponding rat TM sequences with homologies for the forward and reverse primer sequences of 83 and 79%, respectively, amplifying a product of equal size to that of human TM (218bp). The intensity of the band was quantified by densitometric analysis with Scion Image software, and divided by that of the corresponding band of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results were presented as the ratio of TM/GAPDH of IVCs simultaneously taken from rats without any treatment.
2.6. Immunohistochemical analysis
Immunohistochemical staining was performed using an LSAB2 kit (K0609; DAKO, Carpinteria, CA), diaminobenzidine chromogen, and hematoxylin counterstaining. The primary antibodies used were a rabbit anti-human TM polyclonal antibody (H-300; Santa Cruz Biotechnology Inc., CA), and a mouse anti-human von Willebrand factor (VWF) monoclonal antibody (F8/86; DAKO, Carpinteria, CA).
2.7. TM cofactor activity and bound thrombin activity assays
Measurement of TM co-factor activity was performed on veins immediately after harvest or after artificial perfusion. Veins were opened longitudinally. After the end was excised, the remainder was closed inside out by continuous 7-0 sutures or clipping (LT100; Ethicon Inc., Cincinnati, OH), so that only endothelium was exposed on the outside. TM cofactor activity in the endothelial cell surface was determined as described previously [10]. Briefly, exogenous protein C (0.16µM) (Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) was activated by endothelial cells in the presence of thrombin, and cleavage of substrate S2266 (Chromogenix, Stockholm, Sweden) measured. The results were expressed as the changes in optical density at 405nm/min. Measurement of bound thrombin activity was determined essentially as described [14]. Vessels were incubated at 37°C with 333µmol/l of the chromogenic substrate, S-2238 (Chromogenix, Stockholm, Sweden). After 30min, the supernatants were removed and the conversion of the substrate determined spectrophotometrically. Vessel segments were then incubated for 5min with excess lepirudin, washed with PBS, and incubated a second time with S-2238. The difference in absorbance at 405nm before and after lepirudin treatment was taken to represent the thrombin-specific conversion of the substrate. Bound thrombin activity was quantified by comparison to a human thrombin standard curve. Each measurement was performed in five samples and the results expressed as the means±SD.
2.8. Statistics
Mann–Whitney analysis was performed for statistical analysis. A P value less than 0.05 was considered significant.
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3. Results
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3.1. TM expression after transferrin-facilitated gene transfer
TM expression in rats was evaluated by Western blotting analysis in harvested IVCs and other organs 48h after transfection. Trials of gene transfection using variable doses of DNAs revealed that TM expression in the IVC is the most efficient when 7.5µg of TM DNAs is used (Fig. 1). Further increases of TM DNAs up to 100µg augmented TM expression mainly in other downstream organs like the SVC (Fig. 1). Therefore, subsequent TM transfections were performed using 7.5µg of TM DNAs. Analysis of other organs revealed that our anti-human antibody recognized both endogenous and transfected human TM, and that the former was the most abundant in the lungs (Fig. 2). Transferrin-facilitated gene transfer enhanced the expression of TM protein in various organs, although it was most remarkable in the IVC, suggesting the release of liposome:transferrin:TM gene complexes into systemic circulation after the restoration of full blood flow resulted in significant transfection in downstream organs (Fig. 2). RT-PCR using primers detecting human TM revealed a distinct band corresponding to the size of the transfected human TM gene (218bp) in the IVC, lungs, and circulating white blood cells, and at a lower level in the SVC of transfected rats (Fig. 3), which is in accordance with the TM expression shown by Western blotting analysis. Due to the homology with the rat TM sequence, the RT-PCR primers designed to detect human TM also amplified endogenous rat TM, and a band was weakly detected in the lungs of rat transfected by vector alone (Fig. 3).
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Fig. 1. Western blotting analysis of thrombomodulin (TM) expression after variable doses of TM gene transfection. Protein extracts of superior vena cava (SVC) and inferior vena cava (IVC) were obtained from rats, 48h after the transfection of variable doses of the human TM gene. Western blot analysis was performed with the rabbit anti-TM polyclonal antibody.
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Fig. 2. Western blotting analysis of thrombomodulin (TM) expression in rat tissues. Protein extracts of superior vena cava (SVC), inferior vena cava (IVC), white blood cells (blood), and lung were obtained from rats, 48h after the transfection of the human TM gene (7.5µg of DNAs) (Transfection, hatched columns) or of the liposome:transferrin mixture without the TM gene (Vector, dotted columns). Total levels of TM protein are expressed as the ratio of the density of the bands of IVCs simultaneously taken from rats without any treatment. Values are the mean±SD of n=4 vessels.
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Fig. 3. RT-PCR analysis for TM transcripts. Total RNA of superior vena cava (SVC), inferior vena cava (IVC), white blood cells (blood), and lung were obtained for RT-PCR from rats, 48h after the transfection of the human TM gene (7.5µg of DNAs) (Transfection, hatched columns) or of the liposome:transferrin mixture without the TM gene (Vector, dotted columns). Levels of TM gene expression (TM/GAPDH) are expressed as the ratio to TM/GAPDH of IVCs simultaneously taken from rats without any treatment. Values are the mean±SD of n=4 vessels.
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3.2. TM expression in venous endothelium exposed to arterial circulation
Inferior vena cavas transfected with TM gene or vector alone were harvested, placed into the ex vivo circulation circuit with arterial pressure for 90min, and then subjected to immunohistochemical analysis using specific TM and VWF antibodies. The expression levels were compared with non-perfused veins. Exposure to arterial pressure dramatically decreased TM expression in the venous endothelium of the rats transected by vector alone, whereas the integrity of the endothelial lining, visualized by the VWF immunoreactivity, was well preserved (Fig. 4). TM expression in transfected IVCs does not decrease below normal baseline concentrations, whereas the graft treated by vector alone shows a deficiency after arterial circulation (Fig. 4).
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Fig. 4. Immunostaining for TM and von Willebrand factor (VWF) expression on rat IVCs before and after arterial perfusion. Representative photomicrographs of TM and VWF expression in IVCs transfected with TM gene (7.5µg of DNAs) (Transfection) and with vector alone (Vector) before and after arterial perfusion are presented. The excised veins are stained with a rabbit anti-human TM polyclonal and mouse anti-human VWF monoclonal antibodies. Scale bar is 20µm.
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3.3. Prevention of loss of thromboresistance after exposure to arterial circulation
Inferior vena cavas transfected with the TM gene or with vector alone, and IVCs without any treatments were harvested and the activities of TM and thrombin in endothelium evaluated before and after ex vivo arterial perfusion. TM cofactor activities in IVCs treated by vector alone and IVCs without any treatment were similar (1.36±0.39, 1.41±0.25nM/min/cm2), whereas TM gene therapy increased the activity three-fold (4.05±0.74nM/min/cm2) (P<0.01), suggesting a high capacity for thromboresistance (Fig. 5). After exposure to 90min of arterial perfusion, the activity decreased to less than half in both IVCs treated with vector alone and IVCs without treatment (P<0.01), whereas transfected grafts preserved the activity at the level of normal vein endothelium even after perfusion. (1.56±0.41nM/min/cm2) (Fig. 5). Thrombin activity was very low in all groups of IVCs before exposure to arterial perfusion, but showed a steep increase in IVCs treated by vector alone and in IVCs without treatment after perfusion (Fig. 6). In IVCs receiving TM gene therapy, the increase in thrombin activity after the perfusion was remarkably attenuated (P<0.01) (Fig. 6).
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Fig. 5. TM cofactor activity of the rat IVCs before and after exposure to arterial circulation. TM cofactor activities on endothelium were evaluated in IVCs transfected with TM gene (7.5µg of DNAs) (hatched columns) and with vector alone (dotted columns), and IVCs without any treatment (open columns), before and after ex vivo perfusion with arterial pressure (70mmHg) for 90min. *, P<0.01 vs. vector-transfected and control veins. Values are the mean±SD of n=5 vessels.
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Fig. 6. Thrombin activity bound to the endothelium of rat IVCs before and after exposure to arterial circulation. Bound thrombin activities on endothelium were evaluated in IVCs transfected with the TM gene (7.5µg of DNAs) (hatched columns) or with vector alone (dotted columns), and IVCs without any treatment (open columns), before and after ex vivo perfusion with arterial pressure (70mmHg) using diluted blood for 90min. *, P<0.01 vs. vector-transfected and control veins. Values are the mean±SD of n=5 vessels.
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4. Discussion
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The present study presents two important results. First, our protocol of in vivo transfection using ligand-facilitated transfer of cationic liposome:TM gene complexes efficiently induced the overexpression of TM in the endothelium of IVCs of rats prior to the vein grafting operation. This high efficacy of gene transfection is in accordance with our previous experiments, in which an injection of 50–100µg of cationic liposome cDNA complexes into a peripheral vein of rabbits increased the lipoprotein receptor mRNA levels in leukocytes and decreased the plasma cholesterol levels [9]. This high affinity of the transferrin gene complexes attached to adjacent cells allowed us to perform the gene transfection into the vein endothelium under reduced flow conditions without completely interrupting the blood flow. Second, we demonstrated that this enhanced TM activity in the vein endothelium attenuated the extensive loss of graft capacity to activate protein C, thereby decreasing the bound thrombin activity in 90min of ex vivo arterial perfusion. Thrombin activity in the graft endothelium should be kept to a minimum at all times, not only to prevent graft thrombosis, but also to attenuate subsequent intimal hyperplasia [15]. Our protocol of gene therapy to vein grafts appears to cover the most vulnerable period immediately after the grafting operation, thus preventing graft failure. Although further animal studies are needed to demonstrate the exact benefits of this gene therapy on implanted grafts over a longer period, these results imply a possibility of a new gene therapy for the acute phase of vein graft disease.
Saphenous vein graft failure is composed of discrete processes including thrombosis, intimal hyperplasia and atherosclerosis. Between 3 and 12% of saphenous vein grafts occlude within the first month after coronary bypass grafting surgery [1]. Endothelial dysfunction of the grafted vein appears to be the central mechanism facilitating thrombosis in the early stage after an operation. Among the variety of endothelial dysfunctions, loss of TM activity contributes significantly to early thrombosis [2,3]. In contrast, endothelial protein C receptors appear to maintain their activity [4]. In accordance with the loss of TM activity, tissue factor, a potent initiator of thrombin generation, is induced on the surface of endothelium by exposure to arterial shear stress [16], and by inflammatory cytokines [17]. Other mediators could also be involved, such as tissue plasminogen activator, nitric oxide and prostacycline [18]. Endothelial detachment can even easily take place by over-distension of grafted veins [19], together with hypoxia-induced cytokines and/or platelet-activating factor production [20]. In our ex vivo circulation model, endothelial integrity was preserved during 90min of arterial circulation in all IVC specimens examined, which accords with previous reports of the human saphenous vein model in ex vivo circulation [3], and the rabbit model of interposed vein grafts [4]. However, it is also reported that ex vivo circulation using a patient's vein and blood caused extensive detachment of endothelial cells [19]. We assume that in that study, the high levels of activated cytokines and proteases in the blood remained in the heart-lung machine, and that these, together with the high pressure employed in arterial circulation, may have predisposed for endothelial cell injury and extensive endothelial loss [20]. It is important to minimize the detachment of endothelial cells to achieve the maximal contribution of TM gene therapy. However, to prevent early thrombosis of vein grafts, it is also important to preserve the anti-thrombogenic capacity of the endothelial cells remaining in the vein grafts.
The TM/protein C/protein S pathway has been shown to regulate the anti-thrombogenic properties of endothelium, especially under fluid shear stress [21]. TM binds and sequesters circulating thrombin and catalyzes the conversion of protein C to activated protein C, which, in turn, inhibits further thrombin generation. Accordingly, congenital deficiencies of protein C and TM are associated with an increased risk of thrombosis [21]. In contrast, systemic infusion of recombinant human TM inhibits arterial neointimal hyperplasia after balloon injury in rabbits without accompanying any adverse effects [22]. TM gene expression levels in venous endothelium are reported to decrease after the implantation reaching a nadir after one week [23], whereas TM activity shows an immediate drop independently from TM gene down-regulation [3,23]. In the present study, we have successfully enhanced TM protein expression levels upon graft implantation using highly efficient transferrrin receptor-facilitated transfer [9] of the TM gene. Unlike endogenous TM gene which is suppressible by wall stress [23], our TM cDNA construct driven by a cytomegalovirus promoter and enhancer could express TM gene regardless of surgical manipulation and subsequent wall stress. Thus, although TM cofactor activity derived from transfected TM cDNA showed an appreciable drop after perfusion like endogenous TM protein, the levels after perfusion were still sufficiently high to prevent thromboresistance of vein graft. Furthermore, our previous experiments demonstrated that the therapeutic effect induced even by a single intravenous injection of liposome:transferrin:cDNA complex lasted for more than 2 weeks [9]. Although further study is essential to evaluate the longer term efficacy using an in vivo bypass grafting model, our protocol is expected to facilitate improvements in the endothelial thrombogenic properties that lead to early graft disease.
The most efficient gene therapy protocols currently utilized employ adenovirus-mediated gene transfer, which far exceeds other recombinant viral vectors in terms of transfection efficiency. However, viral vectors have the potential to cause fatal inflammatory immune responses, as well as vector-triggered leukemia in recipients [6]. Therefore, recent ex vivo gene transfer protocols have included careful elimination of the viral solution and washing of the transfected vein segments before implantation [4]. Our ligand-facilitated intravenous gene transfer was free from any of the appreciable immune response reactions often associated with viral vectors, and markedly enhanced the in vivo gene delivery efficiency; clearly showing a high level of clinical benefit [9]. In our protocol, we applied temporary clamps to the IVC to reduce blood flow without interrupting the side branches, and filled the inside of the IVC with a solution containing cationic liposome:transferrin:cDNA complexes after blood dilution was obtained by saline injection. This blood dilution together with the reduced blood flow increases the transfection efficacy (data not shown), otherwise the transferrin gene complexes had a high affinity for adjacent cells and mostly attached to adjacent leukocytes immediately after injection [9]. The present results demonstrating the high efficacy of TM transfection have encouraged us to next apply this method to preoperative patients. We expect that in a clinical situation, TM gene transfer to saphenous veins could be performed under the reduced flow circumstances by applying a blood pressure cuff to the upper thigh, as in venography of varices. Our results suggest a possibility of a new gene therapy applicable prior to a coronary bypass operation; one that will target the acute phase of vein graft thrombosis.
An important question remaining unanswered is whether the gene therapy for overexpressing TM prior to vein grafting could reduce intimal hyperplasia over the longer term, as was shown in the experiments of arterial injury [24]. Adenovirus-mediated TM gene transfer into grafted veins performed simultaneously with implantation failed to prevent intimal hyperplasia developing after 42 days [4]. Acute local thrombosis or mechanical injury is known to trigger cytokine and growth factor gene expression that evokes a secondary cytokine and growth factor response [24]. Such a positive feedback loop would amplify and sustain the proliferative response progressively [24]. Since thrombin causes much more pronounced proliferation of smooth muscle cells in saphenous veins [25], in vivo transfection of the TM gene to maximize the expression of TM protein prior to vein grafting may be effective for blocking intimal thickening.
In conclusion, we have demonstrated that our non-viral in vivo gene delivery protocol efficiently transduced the TM gene in the endothelium of rat IVCs prior to vein grafting, and prevented the loss of thromboresistance of vein grafts exposed to ex vivo arterial circulation. The results suggest a possibility of establishing a new gene therapy strategy applicable prior to an operation; one targeted to prevent early vein graft failure in coronary artery bypass operations.
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Acknowledgments
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This study was supported in part by Grants-in-Aid for Scientific Research (B), 14370405 and 15390219 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Footnotes
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Presented at the joint 17th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 11th Annual Meeting of the European Society of Thoracic Surgeons, Vienna, Austria, October 12–15, 2003.
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Abstract
1. Introduction
