Sonoporation with doxorubicin enhances suppression of intimal hyperplasia in a vein graft model

Journal of Surgical Research 124, 312–317 (2005) doi:10.1016/j.jss.2004.11.001

Sonoporation with Doxorubicin Enhances Suppression of Intimal Hyperplasia in a Vein Graft Model Yoshimasa Mizuno, M.D.,1 Hisashi Iwata, M.D., Ph.D., Hisato Takagi, M.D., Ph.D., Shoji Yoshikawa, Ph.D., Yukio Umeda, M.D., Ph.D., Yukihiro Matsuno, M.D., Ph.D., Yoshio Mori, M.D., Ph.D., and Hirofumi Takemura, M.D., Ph.D. Department of Advanced Surgery, Division of Organ Pathobiology, Gifu University School of Medicine, Gifu, Japan Submitted for publication September 10, 2004

Background. The purpose of the present study is to examine whether sonoporation with doxorubicin enhances suppression of intimal hyperplasia (IH) in a vein graft model. Materials and methods. After the administration of 1.5 mg/kg doxorubicin intravenously, the right external jugular vein of six rabbits was exposed at 2 W/cm 2 and 1 MHz of ultrasound for 2 min (Sonoporation group). Tissue doxorubicin concentration was measured. In 48 rabbits, the right common carotid artery was ligated after performing a vein graft bypass. The animals were divided into the following four groups: the C0 group (surgical procedure only); the C0S (sonoporation without doxorubicin); the C1 (doxorubicin administration only); the C1S (sonoporation with doxorubicin). Twentyfour grafts were subjected to Elastic van Gieson staining for morphometric analysis 4 weeks after the operation; others were subjected to TdT-mediated X-dUTP nick end-labeling for detection of apoptic cells and to staining with a monoclonal antibody against the proliferating cell nuclear antigen for assessment of cell proliferation 1 week after. Results. The tissue doxorubicin concentration was significantly higher in the Sonoporation group than in the Control group. Compared with the C0 group, IH was not suppressed in the C1 group but was significantly suppressed in the C1S group. Sonoporation with doxorubicin administration suppressed IH significantly (C1 group versus C1S group: P < 0.05). Cell apoptosis was induced and cell proliferation was suppressed significantly in the C1S group. Conclusions. Sonoporation with doxorubicin sup1

To whom correspondence and reprint requests should be addressed at Department of Advanced Surgery, Division of Organ Pathobiology, Gifu University School of Medicine, 1-1, Yanagido, Gifu 501-1194, Japan. E-mail: [email protected].

0022-4804/05 $30.00 © 2005 Elsevier Inc. All rights reserved.

pressed IH of the vein graft. Sonoporation may be effective in coronary or peripheral revascularization using vein grafts. © 2005 Elsevier Inc. All rights reserved. Key Words: sonoporation; doxorubicin; vein graft model; anastomotic site; intimal hyperplasia; cell apoptosis; cell proliferation. INTRODUCTION

Although the saphenous vein remains a conduit for coronary or peripheral bypass, its patency rates in coronary artery diseases and lower extremity occlusive diseases decrease to approximately 80 and 69% 5 years after the operation, respectively [1, 2, 3]. There have been several studies of intimal hyperplasia (IH) in clinical cases [4, 5] and experimental models [6, 7] that showed that late graft failure is due to stenosis caused by IH associated with proliferative smooth muscle cells [8]. Therefore, the prevention of smooth muscle cell proliferation is a feasible strategy to suppress IH. Although several drugs [8], an external stent [9, 10], gamma radiation [11], and an anastomosis procedure using nonpenetrating vascular clips [12] have been examined in animal models to prevent stenosis caused by IH, none has proved successful in human cases. Drugs that can prevent proliferation of smooth muscle cells are divided into the four following groups: drugs that prevent DNA synthesis or the mitosis of smooth muscle cells; those that inhibit signal transduction; those that suppress the enzyme or growth factor; and those that induce apoptosis. Sirolimus [13], which prevents signal transduction, and paclitaxel [14], which prevents mitosis, have been proven beneficial in clinical cases of eluting stent. Because antiproliferation drugs affect smooth muscle cells directly, the efficacy of their antiproliferative effect on smooth muscle cells is

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higher than that of other drugs that affect enzymes or growth factor. Uwatoku et al. confirmed the efficacy of NK911, which is a core-shell nanoparticle formed through a self-assembly of block copolymer conjugated with doxorubicin, for the prevention of restenosis after balloon injury [15]. Doxorubicin affects the cell cycle of vascular smooth muscle cells directly by inhibiting DNA topoisomerase and some mediums [16 –19]. Although Uwatoku et al. could not prove the induction of cell apoptosis by doxorubicin, it was reported that doxorubicin induces cell apoptosis through the medium of Bak and Bax, which activates the release of cytochrome C [20]. Despite the strong dose-dependent antiproliferative effect of antineoplastic drugs on cultured smooth muscle cells, their dosage is restricted in clinical cases because of their side effects. Recently, there have been several reports regarding the enhancement of drug uptake to the tissue by sonoporation [21–26]. Ultrasound induces to perturb the cell membrane and/or cell wall to render it more permeable to the drug, and enhances endocytosis or other activated uptake of the entire micelle (with the encapsulated drug) [21]. Doxorubicin is one of the drugs whose uptake is enhanced by sonoporation [21, 22]. Nelson et al. showed that doxorubicin with sonoporation reduced tumor size significantly in a rat model [22]. In this study, we investigated whether doxorubicin with sonoporation enhances the suppression of IH in a model of vein bypass graft. MATERIALS AND METHODS Vessel tissue doxorubicin concentration. Six male Japanese white rabbits weighing 2.5 to 3.0 kg (Chubu Kagaku Shizai, Nagoya, Japan) were anesthetized with an intramuscular administration of 50 mg/kg of ketamine hydrochloride. Each rabbit was placed in a supine position, intubated with an endotracheal tube, and mechanically ventilated. Anesthesia was maintained with an intravenous administration of 2.0 mg/kg pentobarbiturate and 0.3 mg/kg pancuronium bromide. The bilateral external jugular veins were carefully exposed and dissected. Two minutes after the intravenous administration of 1.5 mg/kg doxorubicin, 2 cm of the right external jugular vein was isolated using two clamps. The midpoint of the clamped part of the vein was exposed at 2 W/cm 2, and 1 MHz of ultrasound was applied for 2 min using Sonitron 2000 (Rich-Mar, OK, USA). A 6-mm probe was selected and attached to the vein with jelly. Thirty minutes after the above-mentioned procedure, the bilateral external jugular veins were harvested and flushed with saline. The right and left external jugular veins were defined as the Sonoporation group (n ⫽ 6) and Control group (n ⫽ 6), respectively. Then, tissue doxorubicin concentration was measured by high-performance liquid chromatography [27]. The vein graft model. After the afore-mentioned anesthesia and neck sterilization, the exposure and dissection of the right external jugular vein and the common carotid artery were performed. After 1000 units of heparin was administered intravenously, 2 cm of the right external jugular vein was harvested. The right common carotid artery was bypassed with the harvested vein using an 8-O polypropylene suture with end-to-side anastomosis. The common carotid artery was ligated between two anastomotic sites. Specimen processing. The animals were sacrificed with an overdose of pentobarbiturate, and the bypassed graft including the ca-

TABLE 1 The Four Groups

(1) (2) (3) (4)

The The The The

C0 group C0S group C1 group C1S group

Doxorubicin

Sonoporation

Not administered Not administered 1.5 mg/kg 1.5 mg/kg

(⫺) (⫹) (⫺) (⫹)

rotid artery was harvested with perfusion fixation for morphometric and immunohistochemical analyses. The graft was fixed in 10% formalin for 2 days and then embedded in paraffin. Longitudinal sections (4-␮m-thick) were cut and stained. Sonoporation with doxorubicin in the vein graft model. Two minutes after the intravenous administration of doxorubicin, the right common carotid artery was clamped at the proximal and distal ends of the bypass graft. Immediately after clamping, the proximal anastomotic site was exposed at 2 W/cm 2, and 1 MHz of ultrasound was applied for 2 min. The 24 animals were divided randomly into the following four groups (Table 1): 1. The C0 group (n ⫽ 6) underwent the surgical procedure only. 2. The C0S group (n ⫽ 6) underwent the sonoporation procedure without doxorubicin administration. 3. The C1 group (n ⫽ 6) received doxorubicin intravenously at 1.5 mg/kg. 4. The C1S group (n ⫽ 6) underwent the sonoporation procedure with the intravenous administration of doxorubicin at 1.5 mg/kg. These groups were sacrificed 4 weeks after the operation. Then specimens were examined. Morphometric analysis. Serial sections were stained by the Elastic van Gieson method. Histologically, the anastomotic site was identified by the abrupt end of the arterial inner elastic lamina [28]. The intimal thickness of the graft along the inner elastic lamina was measured at five points with 200-␮m intervals from the proximal anastomotic site. The degree of intimal thickening was expressed as I/M (the thickness of intima/the thickness of media) ratio. Then, the average of five I/M ratios in each group was calculated. Immunohistochemical analysis. The 24 animals sacrificed 1 week after the operation were randomly divided into four groups as mentioned above. The serial sections were subjected to TdT-mediated X-dUTP nick end-labeling (TUNEL) for the detection of apoptic cells and to staining with a monoclonal antibody against the proliferating cell nuclear antigen (PCNA) for the assessment of cell proliferation. The TUNEL index or PCNA index was calculated as follows: the number of TUNEL-positive or PCNA-positive cells in the intima was divided by the total number of cells in the intima per high-power field (⫻400). Examination for side effects of doxorubicin. We examined alopecia and measured body weight, arterial blood pressure, heart rate, levels of aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, white blood cell count, RBC count, hemoglobin, hematocrit, and platelet count 7 days after the intravenous administration of 1.5 mg/kg doxorubicin. All of the procedures were performed under sterile conditions. All animal care complied with the “Principles of Laboratory Animal Care” and the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 80-23, revised 1985). Statistical analysis. The statistical analysis for assessment of doxorubicin tissue concentration, and I/M ratio, TUNEL index, or PCNA index was performed by unpaired Student’s t test and ANOVA followed by Bonferroni/Dunn’s post-hoc test, respectively. A value of P ⬍ 0.05 was considered to be statistically significant.

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RESULTS Vessel Tissue Doxorubicin Concentration

The external jugular vein doxorubicin concentration was significantly higher in the Sonoporation group (67.3 ⫾ 61.3 ng/mg) than in the Control group (6.5 ⫾ 2.2 ng/mg) (P ⫽ 0.0355), as shown in Fig. 1. Morphometric Analysis

The anastomotic site defined as the end of the arterial inner elastic lamina was found easily. The sections from the C0, C0S, C1, and C1S groups are shown in Fig. 2. The intimal thickness of the C0 group appeared to be not smaller than that of C0S group. The intimal thickness of the C1S group seemed smaller than that of the C0 group or the C1 group. The I/M ratios of C0, C0S, C1, and C1S groups are shown in Fig. 3. There was no significant difference in I/M ratio between the C0 group and the C0S group. Sonoporation without doxorubicin administration did not suppress IH. Compared with the C0 group, the I/M ratio was not significantly smaller than that of the C1 group but was smaller than the C1S group (P ⫽ 0.0007). There was a significant difference in I/M ratio between the C1 and the C1S groups (P ⫽ 0.0142). Sonoporation with 1.5 mg/kg doxorubicin administration suppressed IH significantly. Immunohistochemical Analysis

Between the C0 group and the C0S group, there was no difference in TUNEL index. Sonoporation only has no effect on cell apoptosis after grafting. Compared with the C0 group, the TUNEL index of the C1 group tended to be high. However, there was no significant difference in the TUNEL index between the C0 group and that of C1 group. The TUNEL index of the C1S group was significantly higher than that of the C0

The doxorubicin external jugular vein tissue concentration

(ng/mg) 160

*

120 80

group (P ⫽ 0.0225) (Fig. 4). Combining sonoporation with doxorubicin administration induced cell apoptosis strongly. There was no difference in PCNA index between the C0 group and the C0S group. PCNA index in the C1 (P ⫽ 0.004) or the C1S (P ⬍ 0.001) group was significantly lower than that in the C0 group (Fig. 5). Cell proliferation was suppressed by doxorubicin administration with or without sonoporation. Side Effects of Doxorubicin

No abnormalities were detected in hemodynamic variables, liver and renal functions, and hematological *

*

1.6 The average I/M ratio

* : P<.05

FIG. 2. Longitudinal sections of samples from the C0, C0S, C1, and C1S group. The arrows indicate the anastomotic site, which was defined as the end of the arterial inner elastic lamina. The red and yellow triangles indicate the surface and bottom of the intima, respectively. The scales show the regions measured the thickness of intima and media.

1.2

* : P<.05

0.8 0.4

40 0

0

Control group (n = 6)

Sonoporation group (n = 6)

FIG. 1. Tissue doxorubicin concentration of intravenous administration at a dose of 1.5 mg/kg. The tissue concentration of doxorubicin was significantly higher in the Sonoporation group than in the Control group. *P ⬍ 0.05.

Doxorubicin (1.5mg/kg) Sonoporation

C0 (-) (-)

C0S

C1

C1S

(-) (+)

(+) (-)

(+) (+)

FIG. 3. Intimal thickness of the C0 group, the C0S group, the C1 group, and the C1S group. Compared with the C0 group, IH was not suppressed in the C1 group but was significantly suppressed in the C1S group. IH was significantly suppressed by sonoporation with doxorubicin administration (C1 group versus C1S group: P ⬍ 0.05).

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(%)

* : P<.05

*

12

TABLE 2 Side Effects of Doxorubicin Administered Intravenously at a Dose of 1.5 mg/kg

10 TUNEL index

8 6 4 2 0

Doxorubicin (1.5mg/kg) Sonoporation

C0 (-) (-)

C0S

C1

C1S

(-) (+)

(+) (-)

(+) (+)

FIG. 4. TUNEL index. Compared with the C0 group, TUNEL index was not significantly higher in the C1 group, but was in the C1S group.

factors. Neither alopecia nor body weight loss was noted (Table 2). No side effects of doxorubicin were noted. DISCUSSION

The saphenous vein graft patency rate for the bypass coronary arterial diseases at 5 years is 81% [2] and that for bypass of lower extremity occlusive diseases at 5 years is 69% [3]. Because it is difficult to obtain an autogenous artery of adequate length and size, the autogenous vein, and in particular the greater saphenous vein, has been established as the best readily available graft [3]. There were several studies regarding late graft failure due to stenosis caused by IH in clinical cases [4, 5] and experimental models [6, 7]. The early biological adaptation of vein grafts may occur in two phases: first primarily proliferative then primarily secretory, and the regulatory mechanisms involved

(%) 12

*

*

10 PCNA index

* : P<.05

8 6 4 2 0

Doxorubicin (1.5mg/kg) Sonoporation

C0 (-) (-)

C0S (-) (+)

C1 (+) (-)

C1S (+) (+)

FIG. 5. PCNA index. Compared with the C0 group, PCNA index was significantly less in the C1 group or the C1S group.

BW (kg) sABP (mmHg) dABP (mmHg) HR (bpm) WBC (/␮l) RBC (⫻10 4/␮l) Hb (g/dl) Ht (%) Plt (⫻10 3/␮l) AST (U/l) ALT (U/l) BUN (mg/dl) Cr (mg/dl) Alopecia

Preadministration (n ⫽ 6)

7 days after administration (n ⫽ 6)

2.65 ⫾ 0.14 107 ⫾ 14 58 ⫾ 6.0 306 ⫾ 37.3 8166 ⫾ 2660 555 ⫾ 50.9 11.5 ⫾ 1.1 38.6 ⫾ 3.9 31.4 ⫾ 13.1 29.3 ⫾ 6.5 34.7 ⫾ 14.7 15.2 ⫾ 2.9 0.83 ⫾ 0.15 (⫺)

2.67 ⫾ 0.22 114 ⫾ 9.6 57 ⫾ 5.2 284 ⫾ 27.2 9217 ⫾ 2124 590 ⫾ 54.0 12.3 ⫾ 1.0 41.6 ⫾ 2.7 28.6 ⫾ 13.2 32.8 ⫾ 5.4 37.3 ⫾ 17.5 14.3 ⫾ 1.4 0.95 ⫾ 0.14 (⫺)

may be a response to injury, tangential stress, and shear stress [7]. Although the cause of IH has been considered to be the proliferation of smooth muscle cells, the actual mechanism underlying this proliferation has not been clarified yet. Some drugs have been reported to be effective in suppressing the IH of vein grafts. However all of these drugs have not been proven to be effective in clinical cases of revascularization using vein grafts [8]. Drugs that can suppress IH caused by the proliferation of smooth muscle cells are divided into four groups: drugs that prevent DNA synthesis or the mitosis of smooth muscle cells; those that inhibit signal transduction; those that suppress enzymes or growth factor; and those that induce apoptosis. It is considered that antiproliferative drugs are the most appropriate for the suppression of IH because of the inhibition of cell cycle, neointimal formation, and induction of cell apoptosis. For example, sirolimus [13] and paclitaxel [14] are available in clinical cases for the suppression of restenosis after stenting. On the other hand, Uwatoku et al. have examined the efficiency of doxorubicin in preventing restenosis after balloon injury in rats. In their report, they used NK911 (core-shell nanoparticle formed through a self-assembly of block copolymer conjugated with doxorubicin). They showed that NK911 suppresses neointimal formation and vascular smooth muscle cell proliferation [15]. Moreover, because of the induction of apoptosis through a sequential and differential activation of Bak and Bax [20], doxorubicin can suppress IH. Compared with all-trans retinoic acid, which Leville et al. reported [29], doxorubicin suppressed cell proliferation and induced cell apoptosis in the intima more strongly. An antiproliferative drug has severe systemic side effects at a dose required to achieve the desired vascular effect. To alleviate such side effects, a polymeric drug delivery system has been reported [30, 31]. Periadventitial-

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controlled administration, which is a polymeric drug delivery system, of verapamil below 1% of the systemic dose effectively inhibits myointimal hyperplasia in vein grafts [30]. This system, however, has a disadvantage in that the matrices persist at the anastomotic site. Recently, it has been reported that sonoporation is an effective method for gene transfection [32, 33], delivery of decoy oligodeoxynucleotides [34], or drug induction [21–26]. It has been investigated whether the uptake of drugs into cells is enhanced when administered in combination with sonoporation [21–26]. Doxorubicin is one of the drugs whose uptake is enhanced by sonoporation [21, 22]. Husseini et al. have shown the efficacy of doxorubicin induction in vitro [21]. Nelson et al. have reported that a significant tumor size reduction was noted for rats receiving 2.67 mg/kg doxorubicin with sonoporation [22]. The mechanism is hypothesized as follows: (1) ultrasound perturbs the cell membrane and/or cell wall to render it more permeable to the drug, and (2) ultrasound enhances endocytosis or other activated the uptake of the entire micelle (with the encapsulated drug) [21]. We chose doxorubicin for two reasons: doxorubicin has the potential to suppress IH, and its is enhanced by sonoporation. In the present study, the ultrasound parameters were set at levels that showed the efficacy of drug induction by sonoporation as reported in the literature [25]. The external jugular vein tissue doxorubicin concentration increased when doxorubicin was administered in combination with sonoporation. Furthermore, IH was suppressed to a greater extent when doxorubicin was administered in combination with sonoporation than when doxorubicin alone was administered. We used the 6-mm probe of Sonitron 2000 with echo jelly. Therefore, the probe had a performance to deliver ultrasonic effect to a 6-mm-wide space of the attached site. We performed sonoporation focusing on the proximal anastomotic site; approximately, the proximal (artery) and distal (vein graft) 3-mm-long areas of the proximal anastomotic site were covered by sonoporation effect. Uwatoku et al. demonstrated similar results in the balloon injury model: the doxorubicin concentration was 4-fold higher and IH was inhibited more effectively in the NK911 group than in the doxorubicin alone group [15]. We used the vein graft model in our study of doxorubicin study, because it was more practical for surgical applications, such as CABG or arteriosclerosis obliterans treatment, than the balloon injury model. NK911 suppresses neointimal formation and the expression of several cytokines that promote vascular smooth muscle cell proliferation [15]. In our study, it was suggested that during the first week after grafting, doxorubicin with sonoporation induced cell apoptosis as Panaretakis et al. reported [20], and doxorubicin suppressed cell proliferation. It is considered that doxorubicin can suppress cell proliferation without sonoporation, but be-

cause of the cell apoptosis induced by doxorubicin was administered with sonoporation during the first week, intimal thickening was suppressed. A high-dose doxorubicin administration has side effects, such as body weight loss, cardiomyopathy, liver or renal dysfunctions, myelopathy, and alopecia [35]. The acute toxicity of doxorubicin appears approximately 1 week after administration and the 50% lethal dose is 6.0 mg/kg in rabbits. Doxorubicin is clinically administered intravenously at 1.0 mg/kg for malignant lymphoma, lung cancer, gastric cancer, gallbladder cancer, bile duct cancer, pancreas cancer, liver cancer, colon cancer, and osteogenic sarcoma. In the present study, high-dose doxorubicin (1.5 mg/kg) is almost equivalent to the clinical dose, but induced no side effects 1 week after its administration. The effects of doxorubicin on the cardiovascular system can be categorized as acute and chronic. Acute effects include tachycardia, hypotension, and various EKG changes. Chronic effects are irreversible cardiomyopathic changes leading to congestive heart failure [36]. The chronic cardiotoxicity of doxorubicin arises in cumulative dose dependently (500 mg/m 2 clinically). We examined only acute toxicity, because we performed the bolus administration of doxorubicin in this protocol. Acute toxicity was not observed (Table 2). It is considered that sonoporation is an excellent drug delivery system without residual matrices in a polymeric system [30, 31]. Furthermore, sonoporation may be applied clinically and easily because attaching the probe to the target organ can be performed after anastomosis in the surgical field. CONCLUSION

Combining sonoporation with doxorubicin administration increased the tissue doxorubicin concentration and suppressed IH of vein grafts significantly. Doxorubicin administration with sonoporation may be effective to prevent the IH of coronary or peripheral revascularization using vein grafts. REFERENCES 1.

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