British Journal of Radiology (2007) 80, 702-707
© 2007 British Institute of Radiology
doi: 10.1259/bjr/18259234
Inhibition of pseudointimal hyperplasia in swine TIPS models: the efficacy of local delivery of paclitaxel using a perforated balloon catheter
S W Park, MD
1
S H Lee, MD
2
C H Kim, MD
3
G S Jeon, MD
4
S J Hong, MD
2
J G Yi, MD
1 and
H J Jeon, MD
1
1 Department of Radiology, Konkuk University Hospital, 2 Departments of Diagnostic Radiology, 3 Pathology, Korea University, College of Medicine, Seoul, 4 Department of Diagnostic Radiology, Ajou University School of Medicine, Suwon, Korea
Correspondence: Professor Sang Woo Park, 4–12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, Republic of Korea. E-mail: psw0224{at}kuh.ac.kr
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Abstract
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The aim of this study was to investigate the efficacy and feasibility of local delivery of paclitaxel to inhibit pseudointimal hyperplasia/intimal hyperplasia in swine transjugular intrahepatic portosystemic shunt (TIPS) models
TIPS were created in seven healthy domestic swine (15–20 kg). Before TIPS stent insertion, we performed a short-term infusion of paclitaxel (treatment group: n = 4) and saline (control group: n = 3) into the TIPS tract using a balloon catheter in which two 0.010 inch holes were created on opposite sides of the balloon. Paclitaxel or saline was given to all animals via the hepatic parenchymal and venous outflow tract. The animals were followed for up to two weeks and then killed. Gross and histological evaluations of the shunts were performed, and the maximum pseudointimal/intimal hyperplasia thicknesses were calculated for each animal
The average infusion time of paclitaxel or saline was 7.6 min (6–9 min). At gross and histological evaluation, considerable pseudointimal hyperplasia had formed in the control group and statistically significant differences were found upon microscopic evaluation in the maximum pseudointimal hyperplasia thickness between the control (2.41 mm, range 1.7–3.16 mm) and animals receiving paclitaxel (0.63 mm, range 0.42–0.98 mm, p<0.05)
Local delivery of paclitaxel at the time of TIPS creation may have been effective in reducing pseudointimal/intimal hyperplasia in swine TIPS models.
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Introduction
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The use of the transjugular intrahepatic portosystemic shunt (TIPS) has been established as an effective and safe procedure to decompress portal venous hypertension and to control acute variceal bleeding. However, numerous large studies have reported shunt dysfunction in up to 85% of patients within 1 year [1–4]. The shunt dysfunction is caused primarily by the proliferation of myofibroblastic tissue (pseudointimal proliferation) followed by stenosis of the stent or the hepatic venous outflow tract. This pseudointimal proliferation results from smooth muscle cell (SMC) proliferation and collagen deposition by these SMCs. Therefore, there have been several reports of efforts of reducing SMC proliferation and collagen deposition to increase primary patency of TIPS [5].
Recently, the stent graft had been used for TIPS creation to mechanically inhibit the growth of pseudointimal/intimal hyperplasia into the TIPS tract mechanically. One of the animal studies by Haskal et al [6] showed better improvement and control of luminal encroachment using a polytetrafluoroethylene (PTFE)-covered stent than the previous study using bare stents. Otal et al [7] and Hausegger et al [8] had used the Viatorr stent graft (W.L. Gore & Associates, Flagstaff, AZ) for TIPS creation. The 12 month primary and secondary patency rates were more than 80% and 100%, respectively. However, we also found some cases of stenosis in the hepatic venous outflow tract and the junction between the non-covered and covered segment of stent graft.
Paclitaxel, a potent antineoplastic drug, has been shown to be a promising agent for the therapy of ovarian, breast and other cancers [9]. Paclitaxel alters the dynamic equilibrium between microtubules and A- and B-tubulin by favouring the formation of abnormally stable microtubules. This leads to the inhibition of cell division and migration, intracellular signalling and protein secretion, which all rely on the rapid and efficient depolymerization of microtubules [10–13]. Therefore, the pharmacological effect of paclitaxel prevents neointimal hyperplasia and it represents an alternative to radiation therapy in respect to the inhibition of neointimal hyperplasia [9, 14]. Additionally, paclitaxel, a hydrophobic compound, and unlike hydrophilic drugs, may remain in and around target arterial tissues for some time after application.
Therefore, we evaluated the efficacy and feasibility of local delivery of paclitaxel to inhibit pseudointimal hyperplasia in swine TIPS models.
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Methods and materials
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Animal models and anaesthesia
This study was approved by the Institutional Review Board. Eight healthy, young domestic swine (15–20 kg) were included. Each was pretreated with 650 mg of aspirin orally the day before the procedure and given no food overnight. TIPSs were performed in all of the swine within 3 days of delivery to our hospital. A preanaesthetic regimen of intramuscular ketamine (20 mg kg–1) and xylazine (2 mg kg–1) was used. After venepuncture of the right ear, anaesthesia was maintained with 1000 ml of normal saline mixed with 10 ml of ketamine.
Drug preparation and local drug delivery device
Paclitaxel (Taxol®; BMS, Montreal, Canada) consisted of 7.0 mmol l–1 paclitaxel dissolved in a lipoid vehicle, a 1:1 mixture of polyethoxylated castor oil and absolute ethanol. For local delivery, paclitaxel was diluted to a final concentration of 7.0 mmol l–1 in a volume of 40 ml of sterile 0.9% NaCl solution [9].
A local drug delivery device was created with a balloon catheter. An 8x40 mm balloon catheter (UltraThin, Boston Scientific, Natick, MA) in which two 0.010 inch holes were created on opposite sides of the balloon using an 0.010 inch guide wire. We performed the test injection with normal saline into the balloon catheter in order to confirm that saline spouted from the balloon via its two holes.
TIPS creation and local drug delivery
Under fluoroscopy guidance, a contrast material (Ultravist; Schering, Berlin, Germany) was injected into the right ear vein through the inserted intravenous (i.v.) line. The right internal jugular vein was then punctured percutaneously with the use of a micropuncture set (Cook, Bloomington, IN) during injection of contrast material. A 0.035 inch guide wire (Terumo, Tokyo, Japan) was placed in the inferior vena cava (IVC) and a 9-F sheath was placed over the wire and advanced to the IVC. Then, an angled catheter (Cook) was introduced through the sheath. The catheter/sheath assembly was moved up along the right wall of the IVC until the catheter tip popped into the entrance to the right hepatic vein. The wire was placed deep into the hepatic vein and then the catheter and sheath were introduced over the wire into the hepatic vein. Then, we performed portal vein access with a puncture needle.
After successful puncture into the portal vein, a 0.035 inch Amplatz Super Stiff wire (Boston Scientific) was advanced into the superior mesenteric vein and 2500 U of heparin was injected intravenously. A test injection was performed to confirm entry into the portal system, and predilatation through the tract was performed with the use of a 6x40 mm angioplasty balloon catheter (Boston Scientific). The distal end of the 9-F sheath was then carefully advanced into the shunt tract. Contrast material was injected to determine the hepatic and portal venous entry sites. An 8 x40 mm angioplasty balloon catheter (Boston Scientific) was placed in the parenchymal tract. Contrast material was infused through a perforated balloon catheter and staining of contrast material into the parenchymal tract was confirmed (Figure 1
). Thereafter, prepared paclitaxel was infused through the perforated balloon catheter while pressure was maintained between 4 and 6 atm from the portal vein wall to the hepatic venous outflow tract. After drug delivery, 10x70 mm Wallstent (Boston Scientific) deployments were performed in the TIPS tract.
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Figure 1. After successful portal vein puncture, contrast material was infused through a perforated balloon catheter and staining of contrast material(arrow) into parenchymal tract was confirmed.
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Each of the control pigs also received normal saline through the perforated balloon catheter. The stent deployment technique was the same as the previously described delivery technique.
Follow-up portal venography
Animals underwent anaesthesia again for venographic follow-up of the TIPS 2 weeks after the procedure. This time point was chosen because an earlier study in the same model demonstrated marked intimal hyperplasia and complete or near-complete shunt occlusion by 2 weeks [15].
Under ultrasound guidance, percutaneous direct portal vein puncture with a micropuncture set was performed. A 5-F angiographic catheter was inserted into the superior mesenteric vein and portal venography was performed. Finally, all swine were killed under deep anaesthesia by intravenous administration of 30% potassium chloride solution (15 ml), and tissue was removed for gross and histological evaluation.
Gross and histological evaluation
Liver tissue surrounding TIPS, the hepatic IVC and the portal vein was carefully removed en bloc at necropsy and immersion fixed in 4% neutral buffered formalin for at least 24 h before the shunts and stents were carefully bisected in a longitudinal fashion. The stent struts were removed by hand only. Macroscopic evaluation was performed to document stent position and patency along with gross surface appearance of the vein covering. All histological sections were stained with haematoxylin and eosin stain. The pseudointimal or intimal hyperplasia thickness was measured for defects caused by the stent struts to the lumen, which was perpendicular to an imaginary line connecting the defects. The three thickest parts were measured and then averaged to obtain the mean maximum pseudointimal/intimal hyperplasia thickness. Two observers performed the measurement. Finally, we analysed the mean maximum pseudointimal/intimal hyperplasia thickness between the control group and the treatment group.
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Statistical analysis
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Data were reported by means of median values with ranges of the mean maximum pseudointimal/intimal hyperplasia thickness between two groups. The interobserver variability of the measurement was assessed by using the intraclass correlation coefficients. The Wilcoxon rank sum test was used for comparing the values of the groups. A level of p<0.05 was considered statistically significant.
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Results
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TIPS creation with the infusion of paclitaxel or normal saline was technically optimal in seven out of eight pigs. One pig in the control group died during portal vein access because the extrahepatic portal vein was punctured. Dilatation of this tract was carried out using the balloon catheter. This was the only case in which the extrahepatic portal vein was punctured; therefore, our analysis was performed with four pigs in the treatment group and three pigs in the control group. The average infusion time of paclitaxel or saline was 7.6 min (6–9 min). Portal venography was performed in four pigs because three pigs had died during ketamine iv dripping and percutaneous portal vein puncture. Two pigs received paclitaxel and two pigs were in the control group. The portal venography showed three pigs to be patent, but one pig in the control group showed complete occlusion of the TIPS tract.
Gross evaluation showed that all of implanted stents showed complete coverage from the portal vein into the IVC. Each of the pigs in the control group experienced almost complete occlusion, seen by the tint of white and yellow tissue, of the liver parenchymal tract and hepatic venous outflow tract. However, the treatment group showed relatively little white tissue occlusion of the TIPS tract and the lumen remained smooth (Figure 2). We measured the three thickest parts of the neointimal hyperplasia and then calculated the mean maximum pseudointimal/intimal hyperplasia thickness. The median value of the mean thickness was 2.41 mm (range 1.7–3.16 mm) in the control group and 0.63 mm (range 0.42–0.98 mm) in the treatment group. Interobserver agreement was excellent with intraclass correlation coefficients of 0.99 (Tables 1 and 2). The treatment group turned out to have less pseudointimal/intimal hyperplasia thickness. A statistical analysis by the Wilcoxon rank sum test resulted in statistically significant differences between the groups in terms of the mean maximum pseudointimal/intimal hyperplasia thickness (p<0.05) (Figure 3).
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Figure 2. Gross specimen of TIPS tract longitudinally bisected included the portal vein(PV), hepatic parenchyma, hepatic vein and inferior vena cava (IVC). (a) Gross specimen of the control group shows an occluded shunt filled with whitish and yellowish tissue (arrows). (b) The treatment group shows little occlusion relatively and a smooth surface in the internal surface of shunt (arrows).
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Table 1. The median values with ranges of the mean maximal thickness of pseudointimal/intimal hyperplasia at three points in the treatment group on microscopic evaluation
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Table 2. The median values with ranges of the mean maximal thickness of pseudointimal/intimal hyperplasia at three points in the control group on microscopic evaluation
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Figure 3. Low-magnification photomicrograph of TIPS tract (magnification x40, haematoxylin and eosin stain). (a) In the control group, from defects caused by stent struts to the lumen, there turned out to be a significant proliferation of fibroblast, which demonstrates pseudointimal hyperplasia (arrow). (b) In the treatment group, there was significantly less proliferation of fibroblast than in the control group (arrow).
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Discussion
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A few large animal models have been used for studying TIPS, including dog and swine with and without portal hypertension. The liver vascular anatomy in dogs is so different from that in humans that creation of a portal-to-hepatic shunt, from the inferior vena cava to the portal vein, is technically very challenging [16]. Liver anatomy in swine mimics that in humans and TIPS can be created using the same technique as that used in humans [17].
The primary patency rates of TIPS with bare stents are between 25% and 85% at 1 year follow-up, with an average of 50% in larger studies. Although secondary patency rates can be obtained by secondary interventions, these results are still not at a satisfactory level [18, 19]. The proliferation of myofibroblastic tissue (pseudointimal hyperplasia) is a major cause of TIPS stenosis in humans. This neointimal hyperplasia results from smooth muscle cell proliferation and collagen deposition [20]. In some reports that have demonstrated bile duct transections, bile extravasation into a TIPS was thought to play a role in the mechanism for pseudointimal hyperplasia [5, 21]. However, Teng et al [5] suggested that the proliferative response in TIPS was not primarily due to bile leak and that bile leak might facilitate thrombosis. Therefore, the origin of smooth muscle cell proliferation in TIPS walls has not yet been identified.
Two recent clinical studies that used the Viatorr stent graft (W.L. Gore & Associates) in TIPS have reported that 12 month primary and secondary patency rates were more than 80% and 100%, respectively. However, it has been proven that there was a slight amount of stenosis in the hepatic venous outflow tract and the junction between the non-covered and covered segments of the stent graft [7, 8].
Siegerstetter et al [22] performed a randomized clinical study comparing standard heparin treatment with a combination of trapidil, a drug with anti-platelet-derived growth factor activity, and ticlopidine, a platelet aggregation inhibitor. Compared with standard heparin treatment, trapidil and ticlopidine with heparin treatment significantly reduced the incidence of shunt stenosis. This was the result of a reduction in the intimal hyperplasia at the hepatic vein. However, this study showed some adverse effects, even if these were mild and reversible; therefore, the treatment had to be withdrawn in 12% of patients, and these treatment regimens may make the patients incompliant. In addition, ticlopidine has the adverse effect of cholestatic liver injury and bone marrow toxicity; therefore, the use of ticlopidine in patients with poor liver function should be considered carefully.
Vascular in-stent restenosis remains a clinical problem and can be expected to increase in incidence as coronary stenting becomes more frequent and is used in less ideal lesions.
Microtubules play an essential role in multiple cellular activities that are relevant to the restenosis process, including cell division, motility, transport and extracellular secretory processes. The mechanism of the action of paclitaxel consists of polymerization of tubulin, which results in the formation of abnormally stable and non-functional microtubules, thereby inhibiting cellular replication in the G0/G1 and G1/M phases. Previous in vitro studies demonstrated inhibition of migration and proliferation of vascular smooth muscle cells by paclitaxel, and initial promising in vivo studies of systemic and local paclitaxel administration to inhibit intimal growth have been reported [10–13, 23].
Unlike other antiproliferative agents, paclitaxel also has several properties that make it a good candidate for local drug therapy of excessive arterial smooth muscle cell proliferation in restenosis after balloon angioplasty or stent implantation; these properties have been tested in vitro [24] in animal models [25] and in clinical studies [26] thus far. First, the highly lipophilic character of paclitaxel promotes rapid cellular uptake by enabling it to pass easily through the hydrophobic barrier of cell membranes [27]. Second, the unique mode of action supports a long-lasting antiproliferative action even after a brief, single-dose application at very low concentrations, as previously shown in tumour cells [28]. An antiproliferative effect of paclitaxel on vascular cells has been shown in vitro in rat vascular smooth muscle cells as well as in vivo in the rat carotid artery injury model [14].
To our knowledge, there have been no reports of the infusion of paclitaxel into the vein and TIPS tract and we tried to perform the infusion of paclitaxel using a perforated balloon catheter. All subjects successfully received the paclitaxel into the parenchymal and venous outflow tract of the shunt and this local delivery of paclitaxel into the TIPS tract demonstrated inhibition of pseudointimal hyperplasia.
There were some limitations to our studies. These include (i) the porcine liver had no portal hypertension, (ii) the relevance to humans of this method was not considered, (iii) the optimal infusion time and velocity of paclitaxel were not standardized, (iv) follow-up portal venography was not performed in all pigs, (v) the delivery of paclitaxel was finished after just one infusion using a perforated balloon catheter and (vi) the number of animals employed in this study was extremely limited.
Despite these limitations, this experimental study suggests that local delivery of paclitaxel using a perforated balloon catheter in a TIPS tract may improve TIPS patency by inhibition of pseudointimal hyperplasia.
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Acknowledgments
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This study was supported by a grant from the Schering Research Fellowship for Diagnostic Radiology of the Korean radiological society.
Received for publication September 28, 2006.
Revision received October 29, 2006.
Accepted for publication November 27, 2006.
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Abstract
Introduction
