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(Radiology. 2001;219:716-723.)
© RSNA, 2001

Experimental Studies

Comparative Trial of Local Pharmacotherapy with L-Arginine, r-Hirudin, and Molsidomine to Reduce Restenosis after Balloon Angioplasty of Stenotic Rabbit Iliac Arteries1

Marc Kalinowski, MD, Heiko Alfke, MD, Sonja Bergen, MS, Klaus Jochen Klose, MD, PhD, James J. Barry, PhD and Hans-Joachim Wagner, MD, PhD

1 From the Department of Diagnostic Radiology, Philipps-University Hospital, Marburg, Germany (M.K., H.A., S.B., K.J.K., H.J.W.); and Boston Scientific, Natick, Mass (J.J.B.). Received August 8, 2000; revision requested September 20; final revision received January 2, 2001; accepted January 11. Address correspondence to H.J.W., Department of Radiology, University of Wisconsin Hospital and Clinics, D4/356 Clinical Science Center, 600 Highland Ave, Madison, WI 53792-3252 (e-mail: hwagner@mail.radiology.wisc.edu). J.J.B. is vice president, Center for Molecular Interventions for Boston Scientific.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To determine if local application of L-arginine, r-hirudin, or molsidomine significantly reduces restenosis after balloon angioplasty in stenotic rabbit iliac arteries.

MATERIALS AND METHODS: Thirty-one male cholesterol-fed New Zealand white rabbits underwent balloon dilation of both common iliac arteries to induce arterial stenosis. Four weeks later, one stenotic iliac artery was simultaneously dilated and received local application of L-arginine (210 mg/mL, n = 7), r-hirudin (0.5 mg/mL, n = 8), or molsidomine (0.2 mg/mL, n = 8) with a channeled balloon catheter. On the contralateral side, 0.9% saline was injected as a control. In eight sham animals, saline was applied to one iliac artery and balloon dilation to only the contralateral artery. Six weeks after local treatment, vessels were harvested, and computerized morphometric and immunohistologic analyses were performed.

RESULTS: Application of drugs resulted in a significant reduction of neointimal area as follows: 53% with L-arginine (1.01 mm2 vs 2.17 mm2, P < .05), 43% with molsidomine (1.04 mm2 vs 1.89 mm2, P < .05), and 20% with r-hirudin (1.79 mm2 vs 2.24 mm2, P < .05). Infusion of saline led to a significant increase (50%, 1.21 mm2 vs 1.93 mm2, P < .05) in neointimal area compared with balloon dilation alone. Immunohistologic findings showed a significant reduction of macrophages (5.0% vs 10.2%, P < .05) and proliferating cells (6.2% vs 10.6%, P < .05) in the neointima after local application of L-arginine.

CONCLUSION: Reduction of neointimal area was significant for L-arginine and molsidomine but not for r-hirudin. Saline infusion caused significant arterial trauma, resulting in additional neointimal proliferation.

Index terms: Arteries, iliac, 984.721 • Arteries, stenosis or obstruction, 984.721 • Arteries, transluminal angioplasty, 984.1279, 984.1282 • Interventional procedures, experimental, 984.1279, 984.1282


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Percutaneous transluminal angioplasty is an established method to treat peripheral occlusive arterial disease. It achieves technical success rates of up to 95%. However, the results are limited by a restenosis rate of 30%–50% in the first 3–6 months in certain vascular areas (eg, femoropopliteal segment) (1). Restenosis is a complex process involving many redundant pathologic conditions, including platelet deposition and thrombus formation, inflammation, smooth muscle cell proliferation, and vascular remodeling.

At this time, the causes of restenosis are not completely understood (2–4). In numerous clinical trials (5–7), systemic application of antiproliferative or antithrombotic compounds has not been proven effective for the prevention of restenosis following angioplasty, although animal studies showed promising results (8–10). One of the main reasons for this phenomenon may be that 10- to 50-fold lower doses of drugs were applied in the clinical studies compared with animal studies. Thus, local delivery of drugs may achieve better results. High drug concentration at the site of arterial injury combined with fewer systemic side effects seem to be a potential therapeutic option to prevent restenosis (11).

Several drug delivery devices are currently being investigated. They allow active drug delivery or passive contact of the substance with the angioplasty region. Side effects of these systems vary due to delivery parameters and composition of the delivered drug. Thus, the safety and effectiveness of the devices must be investigated for each combination of compound and catheter system (12–15).

Hirudin is a specific thrombin inhibitor. Thrombin is generated in large amounts in deep arterial injury and has been shown to induce tissue factor expression in endothelial monocytes and smooth muscle cells, further perpetuating the thrombogenic cycle by initiating the extrinsic coagulation pathway, which results in thrombus generation and its sequelae (16). Thrombin also contributes to smooth muscle cell proliferation by stimulating platelet secretion of growth factors (especially platelet-derived growth factor) and directly acting on smooth muscle cells (17–19).

Arginine is the endogenous nitric oxide precursor and molsidomine, a nitric oxide donor. In addition to their vasodilator effects, nitric oxide plays an important role in vascular regulation through its antiatherogenic and antithrombotic properties. Nitric oxide decreases platelet aggregation and inhibits vascular smooth muscle cell proliferation and migration (20–22). Administration of the nitric oxide precursor L-arginine has been shown (23–25) to restore vascular nitric oxide activity due to hypercholesterolemia or atherosclerosis and is associated with a substantial reduction in intimal thickening.

Most of the currently available animal studies, which investigated local drug delivery for prevention of restenosis after angioplasty, were not comparative and did not use different pharmaceuticals in a standardized animal model and under predefined experimental conditions.

The aim of our particular trial was to compare the effects of some potent antithrombotic and antiproliferative drugs on the primary end point, restenosis. We hypothesized that local drug delivery of r-hirudin, L-arginine, or molsidomine would significantly reduce restenosis after experimental balloon angioplasty in stenotic rabbit iliac arteries.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Stenosis Model
Figure 1 gives an overview of the study design. Thirty-one male New Zealand white rabbits (body weight range, 2.5–3.0 kg) underwent balloon dilation and denudation of both common iliac arteries to induce an arterial stenosis. Prior to intervention, the animals were housed individually, were allowed to acclimate to their cages for 2 weeks, and were started with the 1% cholesterol diet after 1 week and water ad libitum. All animal experiments were performed according to a protocol approved by the local ethical committee on animal research.



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  		Figure 1. Diagram of the study design.

 
Balloon denudation was performed with general anesthesia (ketamine [Ketanest]; Parke-Davis, Berlin, Germany, 50 mg per kilogram of body weight and xylazine [Rompun]; Bayer, Leverkusen, Germany, 5 mg/kg; administered in combination by means of intramuscular injection). To maintain anesthesia, additional doses of ketamine were intravenously administered as required after cannulation of an ear vein. The right carotid artery was exposed, and a 4-F arterial sheath (Terumo, Tokyo, Japan) was introduced. By using fluoroscopic guidance, a standard 0.018-inch guide wire (Boston Scientific, Watertown, Mass) was advanced into the periphery of the common femoral artery. Calibrated intraarterial angiography of the distal aorta and both iliac arteries was performed with the use of a calibrated 0.035-inch guide wire (graduated measuring wire-guide; Cook, Bjaervskov, Denmark) placed in the abdominal aorta. The guide wire has graduated gold markers at its tip in distances of 1 cm. Then, a 2-cm-long, 3-mm-diameter balloon catheter (Viper; Boston Scientific) was introduced into both common iliac arteries. The balloon, which was inflated just distal to the aortic bifurcation, was pulled back 1 cm three times, which resulted in mechanical damage of the treated common iliac arteries. After denudation and dilation, angiography was carried out to depict thrombosis, dissection, or signs of vessel perforation. The carotid artery was ligated after removal of the sheath. To prevent thrombus formation, heparin 100 IU/kg was intravenously administered prior to the intervention. The animals were fed a 1% cholesterol diet and water ad libitum until the end of the study.

Drug Delivery Device
The delivery device has been described in detail elsewhere (26). Briefly, it is a three-lumen over-the-wire catheter (channeled balloon catheter; Boston Scientific) with separate ports for balloon dilation and local drug delivery. The balloon has 18 channels, with one group of 15-µm-diameter pores per channel arranged in a spiral pattern along the entire balloon length. The catheter shaft is 3.4 F. For the purpose of this study, we used a balloon length of 20 mm and a diameter of 2.5 mm.

Balloon Angioplasty and Local Drug Delivery
Balloon dilation and local drug delivery were performed (M.K., S.B., H.J.W.) 4 weeks after stenosis induction, with general anesthesia and sterile conditions. To prevent thrombus formation, 100 IU/kg of heparin was intravenously administered prior to the intervention.

The left carotid artery was exposed and a 4-F arterial sheath was introduced. Calibrated angiography was performed in multiple directions (anteroposterior, 30° left anterior oblique, 30° right anterior oblique) to quantify the degree of stenosis. The target vessel and the kind of drug to be applied were randomly selected from a computer-generated random list prior to the intervention. A 0.018-inch guide wire and a guiding catheter were advanced into the femoral arteries. Then a 0.014-inch guide wire (Guidant, Temecula, Calif) was exchanged. After insertion of the guide wire, the channeled balloon catheter (Boston Scientific) was advanced into the area of previous denudation, and the balloon was inflated. Angioplasty balloon inflation was controlled with a manometer (Encore; Boston Scientific) to secure 8 atm (811 kPa) of pressure during inflation.

Simultaneously, drug administration was done at 4 atm (405 kPa) for 3 minutes through the second port of the channeled balloon catheter. The mean application volume was 5 mL. Seven randomly selected animals were treated with L-arginine (21% L-arginine-hydrochloride; B. Braun, Melsungen, Germany; 210 mg/mL), eight animals received molsidomine (Corvaton; Hoechst Marion Roussel, Bad Soden, Germany; 0.2 mg/mL), and another eight animals received r-hirudin (Refludan; Hoechst Marion Roussel; 0.5 mg/kg). By using the same conditions, saline was administered in the contralateral artery serving as a control. In eight additional control animals, saline was injected on one side, and balloon angioplasty with the channeled balloon without drug application was performed on the other side (sham group).

After the drug delivery procedure, the catheter was removed, and completion angiography was performed to check for vessel patency (ie, thrombosis, dissection, or vessel perforation). The sheath was removed and the carotid artery was ligated. Sequential ligation of both carotid arteries, with an interval of 4 weeks, caused no neurologic deficits in any case due to sufficient collateral flow through the vertebral arteries.

Histologic Preparations
Six weeks after balloon dilation and simultaneous local drug delivery, laparotomy was performed with general anesthesia. The infrarenal aorta was cannulated, and calibrated angiography of the iliac arteries was performed. Then, the animals were sacrificed by using a lethal dose of embutramide, mebezonium iodide, and tetracaine hydrochloride (T61; Hoechst, Frankfurt, Germany). The abdominal aorta and the iliac arteries were excised, perfused with saline, and immersion fixed with 4% paraformaldehyde solution. A 1-cm-long vessel segment of both common iliac arteries starting 1 cm distal to the aortic bifurcation was cut into three segments, and the proximal and distal margins were marked with sutures and embedded in paraffin. The segments were cut and stained with routine methods.

After hematoxylin-eosin and van Gieson staining, the segments were morphometrically analyzed by using a semiautomatic planimetric system. The cross sections (4 µm) were projected onto a digital image analyzer (Quantimed 600; Leica, Wetzlar, Germany). The following borders were highlighted with a trackball: external elastic lamina, internal elastic lamina, and luminal-intimal borders. The luminal area, intimal area (defined as the area under the internal elastic lamina), and medial area (defined as the area between external elastic lamina and internal elastic lamina) were measured. Neointimal area was calculated. All segments were morphometrically analyzed, and in all groups, only the sections with the largest neointimal area identified at morphometry were used for data evaluation. All histomorphologic measurements were performed by skilled observers (M.K., S.B.) blinded to the treatment groups. Three cross sections of each vessel segment were measured. The mean value of the two independent measurements of the two investigators was used for statistical analysis.

In addition, immunohistochemical analysis was performed. A monoclonal antibody specific for rabbit macrophages (RAM 11; Dako, Carpinteria, Calif) or for proliferating cell nuclear antigen (PCNA; Dako) was used to identify the different cell populations. Sections were incubated with the primary antibody, anti–rabbit IgG secondary antibody (biotin conjugate), and avidin peroxidase. The peroxidase was then visualized with chromagen (27). The percentage of stained area, as ascertained at immunohistologic analysis, was calculated with a computer-assisted system (Quantimed 600; Leica). The unstained area was categorized as unclassified cell areas consisting of extracellular matrix and unstained cells.

Statistical Analysis
Data are expressed as the mean plus or minus the SEM. All data were analyzed by using the Kolmogorov-Smirnov test to determine the normal distribution. In the case of a normal distribution, statistical significance was tested by using paired t tests for comparisons between the drug-treated and placebo site. In addition, a one-way analysis of variance was performed to compare differences among the treatment groups. A P value less than .05 was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Stenosis Induction and Drug Delivery
In all animals, balloon dilation and denudation led to significant stenoses identified at calibrated angiography 4 weeks after induction. There were no significant differences in luminal diameter reduction in the corresponding common iliac arteries (Fig 2a). The mean diameter stenosis of the left common iliac artery was 45% ± 3 compared to 47% ± 2 on the right side (P = .035, all angiographic data were analyzed and mean stenosis grades were compared). In all animals, the placement of the channeled balloon catheter and the drug delivery procedure was technically successful (Fig 2b). In one animal, a non–flow-limiting dissection was seen after balloon dilation and local drug delivery. All vessels were patent on the final angiogram (Fig 2c). Acute thrombosis or vessel perforation of the target vessels were not angiographically detected in any animal.



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  		Figure 2a. Series of calibrated anteroposterior intraarterial angiograms. (a) Native rabbit iliac arteries prior to treatment. (b) Four weeks after stenosis induction, there is a similar degree of diameter stenosis in both common iliac arteries (arrows). (c) Placement of the channeled balloon catheter (arrow) in the left common iliac artery. (d) After angioplasty and local drug delivery, an increase in luminal diameter and a postinterventional vessel spasm in the external iliac arteries (arrows) can be noted. (e) Six weeks after percutaneous transluminal angioplasty and local drug delivery, there is bilateral restenosis (arrows).

 


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  		Figure 2b. Series of calibrated anteroposterior intraarterial angiograms. (a) Native rabbit iliac arteries prior to treatment. (b) Four weeks after stenosis induction, there is a similar degree of diameter stenosis in both common iliac arteries (arrows). (c) Placement of the channeled balloon catheter (arrow) in the left common iliac artery. (d) After angioplasty and local drug delivery, an increase in luminal diameter and a postinterventional vessel spasm in the external iliac arteries (arrows) can be noted. (e) Six weeks after percutaneous transluminal angioplasty and local drug delivery, there is bilateral restenosis (arrows).

 


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  		Figure 2c. Series of calibrated anteroposterior intraarterial angiograms. (a) Native rabbit iliac arteries prior to treatment. (b) Four weeks after stenosis induction, there is a similar degree of diameter stenosis in both common iliac arteries (arrows). (c) Placement of the channeled balloon catheter (arrow) in the left common iliac artery. (d) After angioplasty and local drug delivery, an increase in luminal diameter and a postinterventional vessel spasm in the external iliac arteries (arrows) can be noted. (e) Six weeks after percutaneous transluminal angioplasty and local drug delivery, there is bilateral restenosis (arrows).

 


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  		Figure 2d. Series of calibrated anteroposterior intraarterial angiograms. (a) Native rabbit iliac arteries prior to treatment. (b) Four weeks after stenosis induction, there is a similar degree of diameter stenosis in both common iliac arteries (arrows). (c) Placement of the channeled balloon catheter (arrow) in the left common iliac artery. (d) After angioplasty and local drug delivery, an increase in luminal diameter and a postinterventional vessel spasm in the external iliac arteries (arrows) can be noted. (e) Six weeks after percutaneous transluminal angioplasty and local drug delivery, there is bilateral restenosis (arrows).

 


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  		Figure 2e. Series of calibrated anteroposterior intraarterial angiograms. (a) Native rabbit iliac arteries prior to treatment. (b) Four weeks after stenosis induction, there is a similar degree of diameter stenosis in both common iliac arteries (arrows). (c) Placement of the channeled balloon catheter (arrow) in the left common iliac artery. (d) After angioplasty and local drug delivery, an increase in luminal diameter and a postinterventional vessel spasm in the external iliac arteries (arrows) can be noted. (e) Six weeks after percutaneous transluminal angioplasty and local drug delivery, there is bilateral restenosis (arrows).

 
Morphometric Analysis
Neointimal areas of drug-treated and control segments following balloon dilation and local drug delivery are illustrated in Figure 3. In none of the cases was deep arterial injury, defined as rupture of the internal elastic lamina, seen.



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  		Figure 3. Graph demonstrates neointimal area assessed with quantitative morphometry after local application of different drugs. Values are expressed as the mean plus or minus SEM. There is a statistically significant reduction of neointimal area after L-arginine and molsidomine application when compared with control (C). Application of saline resulted in a significant increase in neointimal formation when compared with balloon dilation alone. * = P value less than .05 versus control or sham.

 
Administration of L-arginine resulted in a significant reduction of neointimal area of 53% compared with the contralateral control vessel segment, which received saline with the same conditions (1.01 mm2 ± 0.47 vs 2.17 mm2 ± 0.7, P < .05). r-Hirudin reduced neointimal area by 20% (1.78 mm2 ± 0.6 vs 2.24 mm2 ± 0.6). However, this did not reach statistical significance (P < .05). Local administration of molsidomine resulted in a significant decrease of neointimal area of 45% (1.04 mm2 ± 0.28 vs 1.89 mm2 ± 0.61, P < .05). The contralateral control vessel segment, which received local administration of saline, resulted in a significant increase in neointimal area of 50%, compared with sham vessel segments (1.93 mm2 ± 0.3 vs 1.21 mm2 ± 0.16, P < .05). Figure 4 shows representative cross sections of treated and control vessel segments. The one-way analysis of variance did not reveal a statistically significant result (P > .05).



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  		Figure 4a. Photomicrographs of common iliac arteries in cholesterol-fed rabbits 10 weeks after balloon denudation and 6 weeks after balloon dilation and local drug delivery. Neointimal area (NA) is markedly reduced in vessel segments treated with (a) L-arginine and (b) molsidomine compared with (c) control segments treated with saline by using similar conditions. (d) Local administration of r-hirudin resulted in a nonsignificant reduction of neointimal area. Note the intact internal elastic lamina (arrowhead) in all cases. (Elastica van Gieson stain in a, c, and d; hematoxylin-eosin stain in b; original magnification, x40.)

 


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  		Figure 4b. Photomicrographs of common iliac arteries in cholesterol-fed rabbits 10 weeks after balloon denudation and 6 weeks after balloon dilation and local drug delivery. Neointimal area (NA) is markedly reduced in vessel segments treated with (a) L-arginine and (b) molsidomine compared with (c) control segments treated with saline by using similar conditions. (d) Local administration of r-hirudin resulted in a nonsignificant reduction of neointimal area. Note the intact internal elastic lamina (arrowhead) in all cases. (Elastica van Gieson stain in a, c, and d; hematoxylin-eosin stain in b; original magnification, x40.)

 


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  		Figure 4c. Photomicrographs of common iliac arteries in cholesterol-fed rabbits 10 weeks after balloon denudation and 6 weeks after balloon dilation and local drug delivery. Neointimal area (NA) is markedly reduced in vessel segments treated with (a) L-arginine and (b) molsidomine compared with (c) control segments treated with saline by using similar conditions. (d) Local administration of r-hirudin resulted in a nonsignificant reduction of neointimal area. Note the intact internal elastic lamina (arrowhead) in all cases. (Elastica van Gieson stain in a, c, and d; hematoxylin-eosin stain in b; original magnification, x40.)

 


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  		Figure 4d. Photomicrographs of common iliac arteries in cholesterol-fed rabbits 10 weeks after balloon denudation and 6 weeks after balloon dilation and local drug delivery. Neointimal area (NA) is markedly reduced in vessel segments treated with (a) L-arginine and (b) molsidomine compared with (c) control segments treated with saline by using similar conditions. (d) Local administration of r-hirudin resulted in a nonsignificant reduction of neointimal area. Note the intact internal elastic lamina (arrowhead) in all cases. (Elastica van Gieson stain in a, c, and d; hematoxylin-eosin stain in b; original magnification, x40.)

 
Immunohistochemistry
The Table shows the percentage of the neointimal area that stained positively for macrophages and for proliferating cells. Figure 5 shows representative cross sections of immunohistologic staining for monoclonal antibody specific for rabbit macrophages or for proliferating cell nuclear antigen. Of the neointimal area, 10%-15% was infiltrated by macrophages in the control groups, whereas L-arginine and r-hirudin significantly reduced surface area for positively stained macrophages. Moreover, 4 weeks after denudation and 6 weeks after local drug delivery, proliferating cells were significantly reduced in segments treated with L-arginine and molsidomine compared with control vessel segments.


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  		Percentage of Neointimal Area That Stained Positively for Macrophages and Proliferating Cells

 


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  		Figure 5a. Photomicrographs of iliac artery segments immunohistochemically stained for (a) macrophages (monoclonal antibody specific for rabbit macrophages [RAM 11; Dako]) and (b) proliferating cells (monoclonal antibody specific for proliferating cell nuclear antigen [PCNA; Dako]). Red staining indicates a positive reaction with rabbit macrophages and proliferating cell nuclear antigen. (Original magnification, x40.)

 


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  		Figure 5b. Photomicrographs of iliac artery segments immunohistochemically stained for (a) macrophages (monoclonal antibody specific for rabbit macrophages [RAM 11; Dako]) and (b) proliferating cells (monoclonal antibody specific for proliferating cell nuclear antigen [PCNA; Dako]). Red staining indicates a positive reaction with rabbit macrophages and proliferating cell nuclear antigen. (Original magnification, x40.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
This comparative study showed that a single-dose administration of the nitric oxide donors L-arginine and molsidomine significantly reduced intimal thickening after balloon angioplasty. The specific thrombin inhibitor r-hirudin showed, to a lesser extent, similar effects in reducing restenosis in rabbit iliac arteries. To our knowledge, testing of different pharmaceuticals by using similar conditions in a standardized animal model has not been previously described.

Nitric oxide plays a pivotal role in the restenosis cascade, and it has been previously described (28) that systemic application of L-arginine restores endothelium-dependent vasodilator function in animals and humans. L-arginine enhances nitric oxide synthesis and is associated with reduced endothelial adhesiveness for platelets and monocytes, which results in less cell accumulation in the vessel wall. This effect may be due to cyclic guanosine 3',5'monophosphate–dependent alterations in signaling of adhesion pathways by inhibiting expression of endothelial adhesion molecules and chemokines involved in monocyte and platelet adhesion (20,21,29–33).

Nitric oxide has also been shown (22–24) to suppress the proliferation and migration of vascular smooth muscle cells mediated by cyclic guanosine 3',5'monophosphate–dependent pathways. L-Arginine limits the progression of atherosclerosis. Moreover, nitric oxide is a direct scavenger for superoxide anions, and it has beneficial effects on arterial remodeling (30,34). It was previously demonstrated that long-term oral administration of L-arginine reduces intimal thickening after arterial injury. Hamon et al (24) showed that administration of nitric oxide in animals before denudation could substantially reduce intimal thickening when compared with animals that did not receive L-arginine. Schwarzacher et al (35) reported that the local administration of L-arginine (800 mg) with a Dispatch catheter could enhance vascular nitric oxide generation and inhibit lesion formation in rabbit iliac arteries.

Our results are consistent with the findings of Schwarzacher et al that suggest that these effects were partially associated with the activity of inducible nitric oxide synthase expressed by vascular smooth muscle cells after injury. However, in our study, the dose of L-arginine was 1.3 times higher. In addition, Shimokawa and Vanhoutte (36) reported that neoendothelium has an impaired capacity to synthesize and/or release endothelium-derived relaxing factor (nitric oxide).

Our study demonstrated a significant reduction of neointimal thickening after local administration of molsidomine. Molsidomine is a member of the sydnonimine class of drugs. This is in accordance to findings of Bartoli et al (37), who found a substantial reduction of restenosis after application of 150 mg of molsidomine with a hydrogel-coated balloon in the iliac arteries of minipigs. The sydnonimine ring, a mesoionic heterocycle, opens independently of enzymatic activity and gives rise to a direct nitric oxide–releasing molecule by means of spontaneous degradation. It was previously reported by Groves et al (38) that the active metabolite of sydnonimines reduced platelet adhesion and thrombus formation in a porcine model of balloon angioplasty.

However, in a prospective randomized multicenter trial (Angioplastic Coronaire Corvasal Diltiazem study [39]), administered molsidomine (4 mg orally three times daily for 6 months) and systemically administered linsidomine resulted in only a moderate improvement in long-term angiographic results and showed no effect on clinical outcome. An optimal dose for the underlying animal model is, to our knowledge, not yet known, however, for local drug delivery, we chose a substantially lower dose compared with that used in the study of Bartoli and co-workers (37) and the Angioplastic Coronaire Corvasal Diltiazem study.

In our study, local administration of r-hirudin also had a beneficial effect on intimal thickening after balloon angioplasty with the channeled balloon catheter. However, this effect was not significantly different. Arterial injury activates the coagulation system, generating large amounts of thrombin by means of the formation of the prothrombinase complex and resulting in platelet deposition, thrombus formation, release of vasoactive and mitogenic factors, and migration and proliferation of smooth muscle cells. Thrombin is a potent stimulus for platelet activation and also has mitogenic potential. Moreover, thrombin contributes to smooth muscle cell proliferation by stimulating platelet secretion of growth factors, especially platelet-derived growth factor, and by directly acting on smooth muscle cells (40).

One explanation for the low amount of neointimal reduction after hirudin application may be the fact that we administered a single dose application of 0.5 mg/mL hirudin. Heras and co-workers (41) showed that 0.7 mg/kg administered systemically was the lowest dose that completely inhibited arterial thrombosis in a porcine animal model, whereas 0.3 mg/kg was identified as a suboptimal dose for antithrombotic efficacy. Several studies (18,19,42,43) demonstrated the efficacy of hirudin in preventing mural thrombosis, platelet deposition, and intimal thickening in animal models.

Sarembrock et al (17) described the beneficial effect of a 2-hour infusion of hirudin (0.5–1.0 mg/kg) at the time of angioplasty, which resulted in significantly less restenosis at angiography and histopathologic examination. Local delivery of hirudin (0.7 mg/kg) for 1 hour over a double balloon catheter in swine carotid arteries resulted in a reduction of mural thrombus formation, compared with systemic application. In addition, Thome et al (18) showed that combined early and delayed hirudin administration for 24 hours significantly reduced angiographic restenosis and narrowing of cross-sectional area compared with early or delayed treatment alone.

In contrast to these promising results, the Helvetica study (44) showed no apparent benefit with hirudin compared with heparin in preventing restenosis after angioplasty. In addition, the animals in our study received anticoagulant therapy with heparin to prevent thrombus formation. It is possible that the administered dose was sufficient to suppress the coagulative aspects of neointimal formation without achieving an additional effect by application of r-hirudin.

The aim of local drug delivery is to achieve a high drug concentration in the vessel wall combined with reduced systemic adverse effects. In consequence, the application modalities must be sufficient to achieve a relevant intramural drug deposition. The channeled balloon catheter was designed to allow local drug delivery at low pressure, without jet streams, during simultaneous balloon angioplasty. Safety and effectiveness of the channeled balloon catheter were documented by Hong et al (26), with sufficient penetration of radioactive markers in the inner third of the media at 2 atm (203 kPa) during simultaneous angioplasty at 6 atm (608 kPa).

With our study conditions (4 atm application pressure and 8 atm angioplasty pressure), the procedure resulted in substantial vessel trauma, which resulted in an increase in neointimal formation. Controversial data about the application pressure have been published. Plante et al (45) showed that an application pressure up to 6 atm (608 kPa) with a porous balloon catheter resulted in no additional vessel damage in an animal model. Other studies demonstrated that even lower pressure levels resulted in arterial wall necrosis. Herdeg and co-workers (46) demonstrated that effective local drug delivery could not be achieved with injection pressures less than 2 atm (203 kPa) through a porous balloon catheter. However, the use of higher pressure resulted in disruptive delivery.

In this study, increasing fluid volumes determined the extent of vessel injury. Arterial-balloon ratio, pore size, design of the catheter, and the applied fluid volume will influence the downstream loss of pharmaceutical agents, as well as the pressure and concentration gradients along the vessel wall. Chemical and physical properties of the administered drug (ie, molecular weight, solubility, and electric charge) are other important factors determining penetration of anatomic barriers, such as the internal elastic lamina (47–52). Although histologic examination did not reveal direct signs of a deeper vessel trauma, the additional local drug delivery represented an additional injury in our trial. Nevertheless, local delivery of L-arginine, molsidomine, and r-hirudin resulted in a beneficial long-term result. Thus, limited injury to the vessel wall might be outweighed by positive drug effects if a substantial portion of drug could be delivered into the vessel wall.

Study Limitations
Some limitations have to be addressed. First, pharmacokinetic data of the administered drugs were lacking in this work. Penetration depth, local concentration, and half-life of the pharmaceuticals in the vessel wall was not measured. Therefore, the analysis was reduced to the morphometric evaluation of efficacy and only hypothetical conclusions could be drawn with respect to the benefit of local drug delivery. It would have been interesting to assess and even quantify the extent of cross-sectional mural penetration by using autoradiographic techniques used by others (47,49–51). Second, the animal model limitations must be mentioned. Numerous prior studies have shown that beneficial drug effects on restenosis following balloon angioplasty in the rabbit model could not be duplicated in either primate or human clinical studies. Therefore, the extrapolation of data from an animal model to human atherosclerosis requires caution (53). Reasons for the limited comparability of animal models with humans are the species-related differences, as well as the lack of atherosclerotic disease in treated animal vessels. The major criticism has been the abundant presence of foam cells. However, the atherosclerotic rabbit model we used has been demonstrated to have several factors in common with human atherosclerosis, and its reproducibility has been documented in numerous previous studies. Third, the numbers in the study groups were low and statistical analysis was limited.

Practical application: Restenosis of successfully dilated arterial obstructions is the most important drawback of endovascular therapy for peripheral arterial occlusive disease. Simultaneous local drug application with balloon dilation of arterial obstructions might reduce the process of restenosis formation. These findings demonstrated that the nitric oxide donors L-arginine and molsidomine might be good candidates for use in local drug therapy in humans. However, clinical trials have to be conducted to demonstrate the effect, as shown in animals and in clinical conditions in patients who have atherosclerotic disease.


   ACKNOWLEDGMENTS
 
The authors thank Beate Kleb, RT, Sabine Pankuweit, PhD, and Annette Ramaswamy, MD, for their excellent technical assistance and Peter Zoefel, MSc, for statistical analysis.


   FOOTNOTES
 
Author contributions: Guarantors of integrity of entire study, H.J.W., M.K.; study concepts, H.J.W., M.K., J.J.B.; study design, H.J.W., M.K., H.A.; literature research, M.K.; experimental studies, H.J.W., M.K., S.B., H.A.; data acquisition, M.K., S.B.; data analysis/interpretation, all authors; statistical analysis, M.K., S.B., H.A.; manuscript preparation, M.K.; manuscript definition of intellectual content, M.K., H.J.W., K.J.K., J.J.B.; manuscript editing, H.J.W., M.K., H.A., J.J.B.; manuscript revision/review, J.J.B., H.A., S.B.; manuscript final version approval, all authors.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
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