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Interact CardioVasc Thorac Surg 2009;8:31-34. doi:10.1510/icvts.2008.182329 © 2009 European Association of Cardio-Thoracic Surgery
Adenoviral activin A expression prevents vein graft intimal hyperplasia in a rat modelDepartment of Medical Microbiology and Maastricht Infection Center, University Hospital Maastricht, P. Debyelaan 25, P.O. Box 5800 6202 AZ Maastricht, The Netherlands Received 24 April 2008; received in revised form 14 September 2008; accepted 17 September 2008
*Corresponding author. Department of Cardiothoracic Surgery, St Antonius Hospital, Koekoekslaan 1, P.O. 2500, 3430 EM, Nieuwegein, The Netherlands. Tel.: +31-30-609-2104; fax: +31-30-609-2120.
Autologus vein grafts are used for coronary artery and infra-inguinal bypass procedures. Although initially successful, long-term patency rates are limited by lumen occlusion due to neointima formation by smooth muscle cell hyperplasia. Gene therapy to prevent this smooth muscle cell proliferation has been studied extensively with limited success. Activin A, a member of the transforming growth factor-β super family, promotes the contractile phenotype of smooth muscle cells. Maintaining the contractile phenotype could be a novel strategy to prevent intimal hyperplasia. In an epigastric vein-to-common femoral artery interposition grafts rat model, activin A over-expression resulted in a significant decrease in intimal cross-sectional area and percentage stenosis as compared to the control group. BrdU staining identified lower proliferation rates of the smooth muscle cells in the group treated with activin A. We report for the first time evidence that activin A can diminish vein graft failure in a rat model supporting a novel strategy to prevent intimal hyperplasia.
Key Words: Activin; Hyperplasia; Vein graft; Rat
Over the last 40 years autologus saphenous vein grafts have become popular for coronary artery bypass grafting (CABG) and infra-inguinal bypass procedures in patients with critical limb ischemia. Although the initial results of these procedures are good, recurrence of symptoms due to degeneration and occlusion of the graft occur, this gives rise to 15–30% graft failure within one year and up to 50% of all grafts being occluded 10 years after surgery. Intimal hyperplasia (IH), defined as the accumulation of smooth muscle cells (SMC) and extracellular matrix in the intima of the vein graft, is a major pathology within the first year. Graft occlusion involves the migration of medial vascular smooth muscle cells to the lumen of the graft where they transform into a more synthetic phenotype. These cells start proliferating as well as secreting extra-cellular matrix proteins, resulting in the formation of a neointima [1]. Gene therapy aiming to prevent processes contributing to IH, like smooth muscle cell proliferation, has been extensively studied. However, none of the approaches attempted so far have shown acceptable results. A novel method to reduce IH may be by inducing over-expression of the protein activin A. Activin A belongs to the transforming growth factor-β super family and was initially identified as a protein that induced the release of follicle-stimulating hormone by pituitary cells [2]. Furthermore, it has been demonstrated that this protein is involved in cell-cycle regulation and differentiation of various cell types including endothelial cells, macrophages and SMC [3, 4]. Expression of activin A has been demonstrated in atherosclerotic lesions of hyperlipidaemic rabbits as well as in human atherosclerotic lesions and may change SMC proliferation [5]. Enhanced activin A expression was observed in the rat carotid artery after balloon injury [6]. These findings suggest a role of activin A in atherosclerosis and restenosis, possibly by regulating SMC DNA synthesis. Although activin A has been shown to inhibit the propagation of human endothelial cells, its effect on SMC proliferation is debated [3]. Studies have demonstrated activin A to enhance rat SMC DNA synthesis while others could not confirm this [4, 7]. Engelse and colleagues demonstrated that activin A augments the expression level of SMC-specific markers, indicating that it promotes the contractile phenotype of these cells preventing SMC proliferation [8]. Subsequently they demonstrated the ability of activin A to reduce development of neointimal tissue and even lead to complete absence of intimal hyperplasia in a mouse femoral artery cuff model [9]. Maintaining the contractile SMC phenotype could be a novel strategy in preventing SMC intimal hyperplasia as seen in venous graft failure. In this study we demonstrate the ability of activin A to prevent venous graft failure in an experimental rat model.
2.1. Animal model and surgical procedure Inbred specific pathogen free male Lewis (LEW) rats aged 12 weeks and weighing 250–350 g were obtained (Department of Experimental Animal service, University of Maastricht, the Netherlands. Housing and care of the animals, and all the procedures were approved by the Ethical Committee for the Use of Experimental Animals of the Institution, and were in accordance with the Guidelines for the Care and the Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985). Rats were fed standard rat chow and tap water ad libitum. All surgical procedures were performed under general anesthesia using sterile techniques. Epigastric vein-to-common femoral artery interposition grafts using standard microsurgical techniques were performed. Animals were anesthetized by intraperitoneal (i.p.) administration of sodium pentobarbital (60 mg/kg). An 8-mm segment of ipsilateral epigastric vein was harvested, irrigated with heparinized saline (100 U/ml) and used as a reverse interposition graft to a 3-mm segmental defect of the common femoral artery with eight to ten interrupted 11-0 nylon (Ethicon) sutures. The total ischemic time was >30 min. Activin A-expressing adenovirus (Ad.activin) as well as an adenoviral construct carrying no insert (Ad.mock) were provided by Dr Paul Quax (Gaubius Laboratory, TNO-PG, Leiden, the Netherlands). Adenoviral construction has been previously described by Engelse et al. [9]. In short, human activin A cDNA was inserted into the pCMV adenoviral shuttle vector. The activin A cDNA-containing vector and plasmid pJM17 were cotransfected into HER 9.11 cells, and subsequently viable clones were selected, using a standard purification. Two experimental groups (n=10 animals/group) were used. Ad.activin (1x109 PFU) was injected intravenously via the penile vein immediately after grafting. Controls were injected with Ad.mock (1x109 PFU). 2.3. Histological and morphometrical procedures Three weeks after surgery, a catheter was inserted into the apex of the heart, via which initially physiological salt solution was flushed and then the vessels were perfusion fixed with 3.7% formaldehyde in phosphate-buffered saline, pH 7.4, at physiological pressure (100 mmHg). Vein grafts were removed and fixed overnight for routinely paraffin embedding. Cross-sections (4 µm) were taken for morphometrical analysis and hematoxylin-eosin or Lawson staining (average of three cross-sections per graft). Intimal and medial areas were quantified using a computer assisted morphometry (analySIS®, Soft Imaging System GmbH, Muenster, Germany). The cross-sectional area of the media was defined as the area surrounded by the external and internal elastic lamina. The neointimal cross-sectional area was defined by the area circumscribed by the internal elastic lamina and the arterial lumen. Percentage stenosis was defined as the percentage of cross-sectional area circumscribed by the internal elastic lamina covered by neointima. Final scores were given as mean±S.D. Paraffin-embedded cross-sections (4 µm) were stained with the two-layer indirect immunoperoxidase technique using anti- smooth muscle actin (ASMA, Sigma, Missouri, USA) and anti-BrdU (Sigma, Missouri, USA) monoclonal antibodies (mAb). BrdU was injected intraperitoneally one day before sacrifice. To visualize smooth muscle cells, sections were pre-incubated with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 15 min at room temperature and treated with antigen retrieval buffer. To identify cell proliferation, cross-sections were pre-treated with 3% H2O2 in PBS for 15 min at 37 °C, followed by HCl 2N for 30 min at 37 °C and 0.4% pepsin in 0.01N HCl for 30 min at 37 °C. Monoclonal antibodies were diluted (ASMA 1/1500, anti-BrdU 1/500) in PBS and applied to the slides for 60 min at 37 °C. After three wash steps with PBS for 5 min, a biotinylated goat anti-mouse secondary antibody (1/1200, DAKO Glostrup Denmark) was applied for 30 min at room temperature. Sections were then incubated with alkaline phosphatase-coupled streptavidin (ABC reagent, Vector Laboratories), followed by immunodetection using fast red as a substrate. Finally, sections were counterstained with hematoxylin to visualize nuclei. The presence of positive cells was analyzed by microscopy and scored semi-quantitatively by a blinded observer. Representative cross-sections of the graft were analyzed and proliferation quantified as zero (<10 positive signals per section),  (10–50 positive signals per section), (50–100 positive signals per section), (>100 positive signals per section).
Morphometrical data and cell count numbers are expressed as mean±S.D. Values were compared using the Mann–Whitney U-test and P<0.05 was considered statistically significant.
3.1. Animals and grafts None of the animals had apparent clinical signs of illness. Initial body weight ranged from 250 g to 350 g; final weight ranged from 300 g to 375 g. There was no significant difference in body weight between the experimental groups. One graft failure occurred in each group. Overall graft patency rate was 90%. There were no structural anomalies at the anastomotic sites of the grafts. 3.2. Activin A prevents neointimal hyperplasia All grafted veins developed an increase in medial thickness (80±7 µm) as compared to pre-operative (10±2 µm) and showed neointimal hyperplasia. Activin A administration immediately after vein grafting resulted in a significant decrease in neointimal cross-sectional area and percentage stenosis at three weeks after surgery as compared to the control group (Fig. 1).
3.3. ASMA and BrdU staining ASMA staining showed that the medial and neointimal layer in both activin A-treated and control rats predominantly consisted of smooth muscle cells (Fig. 2a). The BrdU staining showed a significantly lower proliferation rate of smooth muscle cells in the group treated with activin A as compared to the control group. This suggests that the over-expression of activin A prevented excessive proliferation of smooth muscle cells and, therefore, neointimal hyperplasia (Fig. 2b).
Although used for several decades, autologous saphenous vein grafts have a reduced long-term patency as compared to arterial conduits. Saphenous veins are more accessible than arterial conduits, therefore, the introduction of an easy to apply intervention improving long-term patency would be a major advance in cardiac and vascular surgery. Early graft failure occurs within the first months after surgery and is predominantly due to thrombotic events often caused by mechanical trauma during harvesting. Late graft failure tends to occur after several years and is caused by accelerated atherosclerosis. These causes can be managed by antiplatelet and lipid lowering drug therapy which has been proven to be successful [10]. Intermediate vein graft failure is due to neointimal hyperplasia which is most prominent in the first year after surgery but can occur up to three years [11]. Excessive proliferation and migration of several vascular cell types are important components in vascular remodelling and associated pathologies, including angiogenesis, atherosclerosis, and restenosis following percutaneous transluminal coronary angioplasty [12, 13]. In this study we have shown for the first time evidence of the ability of activin A to decrease excessive SMC hyperplasia in a rat model. Activin A is a member of the TGF-β super family, along with transforming growth factor (TGF)-β and bone morphogenetic protein [14]. Although initially identified as a protein that induces the release of follicle-stimulating hormone by pituitary cells, recent studies suggest that this cytokine is also involved in atherogenesis. It has been documented that activin A promotes plaque stability by regulating the expression of scavenger receptor mRNA thereby inhibiting foam cell formation, or by inducing a contractile, non-proliferative smooth muscle cell phenotype in culture [8]. In the rat carotid injury model, activin A expression increased a few hours after injury and activin A immunoreactivity could be found in the developing neointima [6]. Enhanced expression of activin A strongly reduced the formation of neointima in cultured human saphenous vein segments and in mice cuffed femoral arteries [9]. This latter effect of activin A may be of importance as a new therapeutic strategy for improving long-term patency rates. The process of vein graft occlusion involves the transition of vascular smooth muscle cells from a contractile to a more synthetic phenotype. This phenotype is characterized by SMC proliferation and secretion of various extra-cellular matrix proteins ultimately resulting in the formation of neointima. Thus, maintaining SMC in a more contractile phenotype by over-expressing activin A may be an attractive strategy to prevent neointimal hyperplasia. Systemic adenoviral infection mainly targets hepatic tissue resulting in high levels of expression of activin A mRNA and subsequently protein synthesis in the liver and secretion into the bloodstream. We were able to demonstrate that in the vein grafts of rats, injected with an activin A expressing adenovirus, smooth muscle cell proliferation was significantly diminished. More importantly, neointimal cross-sectional area and percentage stenosis were significantly reduced in activin A treated rats. These data confirm that activin A plays a role in controlling smooth muscle cell differentiation and proliferation. Our results suggest that activin A administration in the early postoperative phase may be an attractive new therapeutic modality for preventing neointimal formation and subsequent vein graft failure. Despite the reported beneficial effects in cardiovascular disease, a drawback is the reported evidence for the role of activin A in inflammation, tissue repair and fibrosis [15]. The findings that activin A may promote liver, kidney and lung fibrosis when abundantly present, suggests that we should show restraint in clinical therapeutic applications. It is generally assumed that the major target for adenoviruses is the hepatic tissue. This results in high expression mRNA levels and protein in the liver as well as enhanced plasma concentration. Although Engelse et al. demonstrated that the adenovirus used in the present study had indeed these characteristics in mice, we did not examine the presence over time of the protein in either plasma, graft or liver tissue [9]. Therefore, the strength of evidence that the observed effect can be fully attributed to enhanced activin A levels is limited. Additional experiments are required before the observed effects can be entirely accredited to an increased production of activin A. Future experiments could include simultaneous injections with follistatin, which sequesters activin A and thus inhibits its biological activity and should prevent the beneficial effects of activin A on intima hyperplasia.In conclusion, we have shown for the first time that activin A can decrease vein graft failure in a rat model by decreasing IH. However, due to the possible side effects of activin A, in particular tissue fibrosis, additional studies are required to further determine the clinical therapeutic potential of this protein.
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Abstract
1. Introduction
