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Eur J Cardiothorac Surg 2002;22:266-270
© 2002 Elsevier Science NL

Surgical injury of rat arteries: genetic control of the remodelling process

^a Department of Cardio-Thoracic Sciences, Second University of Naples, via Aquila 144, 80143 Naples, Italy
^b Department of Experimental Medicine, Second University of Naples, Naples, Italy

Received 20 September 2001; received in revised form 19 April 2002; accepted 29 April 2002.

* Corresponding author. Tel.: +39-081-5539035; fax: +39-081-5536350
e-mail: arenzul{at}tin.it

	Abstract

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

 
Objectives: Remodelling and restenosis are complex biological processes responsible for bypass and percutaneous transluminal coronary angioplasty failures which are likely to affect many hundreds of genes. We evaluated the effectiveness of topically applied antisense oligonucleotides in reducing the translation of the messenger RNA for the transcription factor c-myc and in reducing stenosis. Methods: Surgery was performed under sterile conditions; 60 Wistar–Kyoto male rats were anaesthetized by ketamine. The carotid arteries were isolated through a median incision in the anterior neck region. At the same point, 0.5 mm longitudinal incisions were performed. Haemostasis was obtained by an adventitial 8.0 stitch. Thirty animals were given 150 µg of c-myc antisense oligonucleotide (Group A) while the other 30 animals received 150 µg of c-myc control sense oligonucleotide (Group B). Oligo molecules were locally applied through 100 µl of 20% pluronic gel. Rats were sacrificed at 30 days; carotid arteries were explanted and stained. Qualitative histological analysis was performed in all cases; serial sections were made every 25 µ in seven consecutive rats for each group. Morphometric analysis was also performed, luminal and medial area values recorded and the ratio between the two areas calculated. Data from each animal were compared with the corresponding controlateral carotid artery and expressed as mean±standard deviation. Statistical comparison between the two groups was carried out by one-way ANOVA text. Results: Qualitative histological analysis showed marked remodelling with complete disarray of vessel wall, neointima accumulation and evidence of elastic fibres in the adventitia of all animals of Group B versus Group A. Morphometric analysis showed a significant reduction in the lumen area in Group A animals together with increased values of the medial area versus Group B animals. In addition, the ratio between the lumen and medial area was significantly higher in Group A than in Group B (2.61±0.18 versus 1.14±0.33, P<0.0001). Conclusions: c-myc antisense oligonucleotides applied intraoperatively can reduce post-operative stenosis.

Key Words: Stenosis • Remodelling • c-myc • Antisense oligonucleotides

	1. Introduction

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

 
Coronary artery disease is still one of the main causes of death in the Western World. Interventional cardiology with percutaneous transluminal coronary angioplasty (PTCA) and cardiovascular surgery with coronary artery bypass graft (CABG) have considerably improved the quality and quantity of life in patients with myocardial ischaemia [1]. Nevertheless, restenosis is an important complication after revascularization [2–5], in particular after catheter-based interventions.

Restenosis following arterial injury consists mainly of cellular proliferation [6], which may ultimately lead to vascular occlusion. Endothelial lesions, in fact, trigger intense proliferative signals that affect subintimal smooth muscle cells (SMCs) via autocrine and paracrine mechanisms, stimulating their re-entry into the cell cycle. Besides SMCs proliferation, other processes causing restenosis after arterial injury are (a) accumulation of extracellular matrix, composed of tenascin, collagen, fibronectin and proteoglycans, (b) migration of SMCs from media to intima, (c) remodelling, and (d) thrombus deposition at the injury site. The mechanisms leading to such complications are still unknown.

Molecular biology approaches such as gene therapy [7] and antisense oligonucleotide [8] have been recently tested in order to prevent and/or treat remodelling. However, this experimentation has not yet been transposed to clinical practice. All the molecular biology techniques mentioned are designed to control the expression of genes involved in cell proliferation and apoptosis.

One of these target genes is the proto-oncogene c-myc, which is quickly induced in cells after arterial injury and by proliferative signals in general. This gene codes for a transcriptional factor actively involved in the early steps of the passage from G₁ to S phase of the cell cycle.

Several animal models have been used to study the stenosis process and/or test drug effectiveness. Balloon angioplasty [7] has been the most widely used technique to induce stenosis in the arteries of pigs, dogs, rats and rabbits. In this procedure, endothelial cells are damaged or removed by a transcatheter balloon inflated with pressure-controlled air. Vein-to-artery grafts [9] have also been utilized. Potential drugs have been investigated also in in vitro systems [10]. One limit of these models is that balloon angioplasty does not fully mimic the vascular injury occurring during cardiovascular surgery, while the vein-to-artery grafts surgical models require larger animals (pigs, dogs) or are not fully reproducible [7,9,10].

The aim of this investigation is to establish a new model of surgical injury which is simpler and less expensive than models already reported in the literature, and which can induce vascular remodelling and stenosis as well as to use this new model to test the effectiveness of phosphorothioate antisense oligonucleotides targeted against the c-Myc mRNA.

	2. Materials and methods

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

 
2.1. Animals
Studies were carried out on adult Wistar–Kyoto rats whose body weights ranged between 300 and 350 g (La Palma, Italy). The animals were housed in air-conditioned rooms, acclimatized and quarantined for at least 1 week at constant temperature (21±1 °C) and relative humidity of 60% under a regular light/dark schedule (light from 07:00 to 19:00 h), and with free access to food and water.

Human care and proper analgesic anaesthetic and tranquillizing drugs were provided to all experimental animals when needed. The ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Research Council (C.N.R.) in 1996 were followed. The proposed study was approved by the Animal Care and Use Committee of the Second University of Naples.

2.2. Phosphorothioate oligonucleotides
Phosphorothioate antisense and control sense oligodeoxynucleotides have been produced by Genset Oligos (Paris, France) and their sequences are as reported by Bennett et al. [17].

2.3. Vascular injury
Rats were anaesthetized with an intraperitoneal injection of ketamine hydrochloride at a final concentration of 8 mg/100 g of body weight. Anaesthesia was followed by a single dose of ticarcillin antibiotics by means of an intraperitoneal injection (5 mg/100 g of body weight). Sterile techniques were employed at all times during the procedure. Operative field magnification was obtained with loops 3.5x. Hairs in the neck region were trimmed and skin was prepped with polyvinylpyrrolidone iodine.

A median incision of 4 cm in length was performed in the anterior neck region. Neck muscles were shifted laterally and the trachea was identified. The dissection was completed by moving laterally to isolate the left carotid artery, which was then encircled by two 5.0 silk sutures. A plastic atraumatic clamp, usually used in coronary surgery, was applied on the carotid for 10 min to determine a crushing lesion. At the same point where the clamp was applied, a longitudinal incision of 0.5 mm in length was made through the full thickness of the carotid wall using a number 15 blade. Haemostasis was obtained either with a single adventitial 8.0 polypropylene stitch, which was used afterwards as a reference frame for experimental investigation, or through the gentle compression of the artery with cotton swabs, or with a 2 mm² oxyl-methyl-cellulose piece.

Once bleeding stopped, the carotid artery was carefully examined and blood pulsation was checked distally with respect to the incision. In each rat 100 µl of 20% pluronic gel F127 (Sigma Aldrich, USA) containing 150 µg of antisense (Group A) or control sense (Group B) c-myc oligonucleotides was applied on the periadventitial side of the injured artery. Pluronic poloxamer gel remains liquid at 4 °C but rapidly solidifies at 37 °C, when in contact with living tissues. The gel is hydrophilic and rapidly degrades in an aqueous medium. Oligonucleotide release from the pluronic gel is assumed to be completed within a few hours. Skin was then reapproximated by reabsorbable suture and the animals were allowed to wake up and feed early after the procedure.

2.4. Histological analysis
Thirty days after surgical injury, the animals were anaesthetized with an intraperitoneal injection of ketamine hydrochloride and their carotid arteries dissected free from the surrounding tissues, washed with physiological solution and perfusion-fixed with Bouin's fluid. After perfusion, a 3 mm part of both carotid arteries (both left surgically treated and right control arteries of the same rat) was removed and bathed in Bouin's fluid for approximately 18 h. The specimens were then dehydrated through a graded ethanol series, embedded in paraffin and serially transversally cut. Cross-sections (6 µm) were stained with hemallume–eosin.

Image screening and photography were performed using an image analysis system (Zeiss Axioscope light microscope, a ProgRes 3008 colour videocamera and personal computer).

All untreated carotid arteries were preliminarily examined in order to exclude animals presenting arteries with primitive vascular diseases. All specimens (n=60) were qualitatively analyzed in a blind manner by two different pathologists at different times.

Preliminary observations were performed under light microscope at 20x magnification in order to select the cross-sections of treated carotids showing differences in wall thickness and/or vessel structure with respect to control carotids. Detailed morphometrical analysis was performed on ten sections close to the arterial injury site in seven carotids for each animal group using the KS300 image analysis software.

Lumen and medial areas were measured and the ratio between the lumen to media area was calculated. The lumen area was defined as the area enclosed by the internal elastic lamina, while the media area was defined as the area enclosed between the external and internal elastic laminae.

2.5. Statistical analysis
Data were expressed as the mean±standard deviation. Statistical comparison between the two groups as well as between each group and the normal parameter values was carried out by means of one-way ANOVA text. A P value less than 0.05 was considered statistically significant.

	3. Results

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

 
Among the 70 operated rats, three died perioperatively, four had primary atherosclerotic lesions in the carotid arteries, and three had signs of wound infection at the time of the explant. The remaining 60 animals were divided into two groups (A and B) comprising 30 animals, both of which were used for this investigation.

The qualitative analysis showed animal minimal growth or the complete absence of neointimal formation and well-preserved vasal cytoarchitecture, including the adventitia layer and elastic laminae in all of Group A. No carotid artery showed a lumen stenosis higher than 25% (Fig. 1A) .

View larger version (63K): [in this window] [in a new window]   Fig. 1. The histological analysis shows the absence of lumen stenosis and media proliferation in a carotid of Group A (c-Myc antisense oligodeoxynucleotides). Conversely, a significant lumen stenosis >75% together with media thickening can be observed in a carotid of Group B (c-Myc sense oligodeoxynucleotides).

Morphometric analysis highlighted a lumen area significantly higher (35745.4±4204.9 µm²) in Group A in comparison to Group B animals treated with control sense oligonucleotides (23436.8±5920.5 µm²) (P<0.001).

Morphometric analysis confirmed that intraoperative treatment with oligos anti c-Myc is effective in provoking a statistically significant reduction in the medial area (P<0.001) in Group A (13767.2±1972.8 µm²) in comparison to Group B (21109.4±3446.6 µm²) animals (Fig. 2) .

View larger version (75K): [in this window] [in a new window]   Fig. 2. The graphic shows a significantly greater lumen area and lower media area in Group A (c-Myc antisense oligodeoxynucleotides) compared to Group B (c-Myc sense oligodeoxynucleotides).

Morphometric analysis of untreated carotid arteries showed a mean value of the lumen area of 40525.6±3900.4µm² and a mean value of the media area of 14200.5±1900.6 µm².

	4. Discussion

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

 
In the past few years, the progress of molecular biology and the determination of nucleotide gene sequences has resulted in new approaches for studying biological functions and developing new diagnostic and therapeutic strategies. The identification of genes involved in cardiovascular diseases paves the way for the possibility of targeting genes as a potential approach to treat such pathophysiological phenomena.

One of the most exciting advances has been the development of the oligonucleotide antisense technology, which may prove a new strategy for future drugs that make it possible to decrease with high specificity the synthesis of any protein of known gene sequence, either reducing the concentration of the corresponding mRNA molecule via RNAse H or by physically interfering with mRNA translation by polyribosomes [11]. Antisense technology, which allows the gene phenotype to be modified, has been widely used in dissecting complex cellular mechanisms, also including cardiovascular functions [12].

In this paper, we describe in detail the surgical model system we recently developed [13,14] to induce stenosis in rat common carotid arteries, and we also report the morphological data on the effect that phosphorothioated oligonucleotides targeted against the c-myc gene have on controlling stenosis progression when topically added to injured carotids. Papers focused on balloon induced restenosis in rat usually analyze carotids 15 days after intervention [15] arguing that rat metabolism and the cell proliferation rate largely exceed human values. Since the surgical approach, which involves all three tunica layers, is far more traumatic than balloon angioplasty, we choose to analyze rat carotids 1 month after surgery.

Moreover, our procedure makes it possible to identify exactly the cross-sections where the surgical injury has been performed because of the presence of the polypropylene adventitial stitch where remodelling and proliferative phenomena are to be investigated. Conversely the PTCA technique, where the air inflated balloon removes the arterial endothelium all along the vessel, makes it necessary to examine the whole artery in order to identify the region where the damage has been maximal both to evaluate drug effectiveness and to analyze the induced pathophysiological phenomena.

Carotid surgery induces the development of marked stenosis, as demonstrated by the accumulation of extracellular matrix and elastic laminae as well as the proliferation and migration of SMCs. Image analysis of serial cross-sections along all the injured carotid shows that the effects of damage are strictly localized at the injury site.

Histological analysis of rat carotid injured vessels shows that SMCs migrate from the media to the intimal layer. These findings are in accordance with Schwartz's observations [16] on restenosis induced by angioplasty, although we observed a greater variety of reactions in injured vessel walls, presumably because our technique is more invasive than PTCA. Cross-section analysis indicates that stenosis induced by our technique is mostly due to negative remodelling of the vessel rather than to intima hyperplasia.

Our model has been used to test the effectiveness of topically applied phosphorothioate oligonucleotides targeted against the c-myc gene in controlling stenosis progression.

The same molecules have already been tested by Bennett [17] in rat carotid angioplasty and by Mannion [9] in pig vein grafts, respectively. The results indicate that decreasing the intracellular concentration of the c-myc gene product ultimately results in controlling stenosis process.

We had previously determined [13] the pattern of expression of the c-myc gene mRNA which follows surgical injury by semi-quantitative RT-PCR and demonstrated that c-Myc mRNA is induced by surgical injury in our model system and reaches its highest level 4 h later before rapidly decreasing to basal levels at 24–48 h. This finding, along with the central role that c-myc plays in cell proliferation, makes this gene a good target for phosphorothioate antisense technology.

The peak of c-Myc mRNA observed 4 h after surgical injury is slightly delayed in comparison to other studies [18]. This result may be related to the difference in vessels under examination (aorta, coronary or carotid), to the different animal model (rat, dog or pig) and, above all, to the different kind of injury (balloon angioplasty with respect to the surgical injury we performed).

Cell proliferation is not the most important process in restenosis [19,20]. Different studies have shown that lumen size reduction is mainly due to negative remodelling, a process characterized by adventitial fibrosis, matrix accumulation, mural thrombus reorganization and cell migration. In particular, some authors [2] observed that fibroblasts in adventitia differentiate into myofibroblasts which are the cells responsible for matrix accumulation and vessel contraction. Consequently, we suggest that morphometric analysis should be based more on the assessment of medial and lumen areas and on the calculation of their ratio instead of linear measures such as the radius or diameter.

Basically we report evidence that also in our model intraoperative treatment with phosphorothioate c-myc antisense oligonucleotides significantly reduces cell proliferation, inflammatory cell infiltration and medial oedema 30 days after injury.

The sustained effectiveness of c-myc antisense oligonucleotides observed 1 month after surgery may be due to the change in cellular commitment at the time of injury because of the antisense induced decrease of the MYC protein level.

The present study indicates that the model of surgical injury on the common rat carotid is effective in triggering all the phenomena involved in the restenotic process. The model we described can be easily reproduced and is simpler and less expensive when compared to balloon angioplasty or to the venous grafting technique.

In the coming years microarray techniques will elucidate the gene pattern involved in cardiovascular system physiology and consequently allow the development of combined antisense oligonucleotide therapy to prevent post-operative stenosis.

	Acknowledgments

 
The research project is the result of a Doctorate (PhD) in Cardiothoracic Sciences, Second University of Naples, by Murst/CNR "Biomolecole per la Salute Umana" L.95/95, by POP 1994/1999 annualità 1997 and by Regione Campania, L. 41, "DNA Antisenso e Farmaci Antiipertensivi".

	Footnotes

 
Presented at the joint 15th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 9th Annual Meeting of the European Society of Thoracic Surgeons, Lisbon, Portugal, September 16–19, 2001.

	References

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