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(Circulation. 1997;95:1863-1869.)
© 1997 American Heart Association, Inc.

Articles

Local Intramural Delivery of L-Arginine Enhances Nitric Oxide Generation and Inhibits Lesion Formation After Balloon Angioplasty

Severin P. Schwarzacher, MD; Tai T. Lim, MD; Bingyin Wang, MD, PhD; Robert S. Kernoff; Josef Niebauer, MD; John P. Cooke, MD, PhD; Alan C. Yeung, MD

From the Division of Cardiovascular Medicine, Stanford (Calif) University School of Medicine.

Correspondence to Alan C. Yeung, MD, Stanford University School of Medicine, Division of Cardiovascular Medicine, 300 Pasteur Dr, Stanford, CA 94305.

	Abstract

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Background Long-term oral administration of L-arginine has been shown to enhance production of nitric oxide (NO) and to reduce lesion formation. The goal of this study was to determine whether local intramural administration of L-arginine could enhance NO generation and reduce intimal thickening.

Methods and Results New Zealand White rabbits (n=27) received a 1% cholesterol diet. For the short-term study, after 1 week of diet, both iliac arteries were balloon injured. Four weeks later, vasoreactivity was assessed angiographically during infusion of acetylcholine (Ach) before and after delivery of L-arginine or saline into the right or left iliac artery (800 mg/5 mL; 0.2 mL/min, 15 minutes) by use of a local drug-delivery balloon. Vessels were then harvested for measurements of NO. For the long-term study, after balloon injury, drugs were delivered as above into the iliac arteries. Two and 4 weeks after L-arginine delivery, vasoreactivity was determined. Subsequently, the iliac arteries were harvested for histomorphometric analysis and measurements of NO. In the short-term study, local delivery of L-arginine restored endothelium-dependent vasodilatation (Ach 10^-5 mol/L; L-arginine +35±10%; saline -14±5%; P<.001) and enhanced local production of nitrogen oxides (L-arginine 152±28; saline 78±12 nmol/L per milligram of tissue per hour; P<.04). In the long-term study, local administration of L-arginine enhanced vascular NO production as long as 1 week after the injury (L-arginine 394.4±141.6; saline 86.3±34.3 nmol/L per milligram of tissue per hour; P<.01) and reduced intimal thickening 4 weeks later (intima/media ratio: L-arginine 0.56±0.1; saline 1.40±0.2; P<.001), largely due to suppression of macrophage accumulation.

Conclusions A single intramural administration of L-arginine enhances vascular NO generation and inhibits lesion formation. Local augmentation of NO production at the site of balloon angioplasty may be a novel strategy to prevent restenosis.

Key Words: endothelium-derived factors • l-arginine • restenosis • macrophages

	Introduction

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The long-term benefit of coronary balloon angioplasty and atherectomy is limited by the considerably high occurrence of restenosis (40% to 50%) 3 to 6 months after the procedure.¹ Restenosis is in part due to myointimal hyperplasia, a process characterized by vascular smooth muscle cell migration and proliferation.^{2 3} A decrease in total vessel area termed "negative remodeling" or shrinkage has recently been found to contribute to the luminal encroachment.^{4 5} Medical therapies to prevent smooth muscle cell proliferation have been uniformly unsuccessful whereas vascular stenting to obtain optimal lumen gain and inhibition of remodeling has modestly reduced the restenosis rate.^{6 7 8} However, intimal thickening still plays a significant role in stent restenosis.

The architecture of the vessel wall is remodeled in response to changes in the balance of paracrine factors that regulate the proliferation and biosynthetic activity of vascular cells. One of the substances that participates in vascular homeostasis is NO.^{9 10} NO is synthesized from the amino acid L-arginine by NO synthase.¹¹ In addition to relaxing smooth muscle cells, NO inhibits their proliferation.¹² Furthermore, NO inhibits the interaction of circulating blood elements with the vessel wall,^{13 14 15} and its activity is reduced in hypercholesterolemia and after vascular injury.^{16 17 18 19} Administration of the NO precursor (L-arginine) has been shown to restore vascular NO activity in animals and in humans with endothelial vasodilator dysfunction due to hypercholesterolemia or atherosclerosis.^{20 21 22 23 24 25} Long-term enhancement of NO activity (by oral administration of L-arginine) is associated with a significant reduction in intimal thickening due to hypercholesterolemia and/or vascular injury.^{26 27 28 29} However, oral administration of L-arginine has systemic effects (eg, growth hormone release), which may confound interpretation of these studies. Therefore, the present study was designed to determine if intramural administration of L-arginine could locally enhance NO activity and modulate vascular structure in the absence of systemic effects.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Twenty-seven male New Zealand White rabbits weighing 3.8±1.5 kg were entered into the study after a 1-week period of acclimation in the housing facilities of the Stanford University Department of Comparative Medicine. All animals were inspected before the study by a veterinarian and monitored daily by technicians and investigators. The experimental protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. Animals were then fed a high-cholesterol diet (1% cholesterol, Dyets) for 5 weeks. Two protocols (short- and long-term studies) were performed as follows.

Acute Study (n=13)
This study was performed to determine if intramural administration of L-arginine could enhance elaboration of endothelium-derived NO in the short term (Fig 1A).

View larger version (25K): [in this window] [in a new window]   Figure 1. Diagrams illustrating the experimental protocols of the short-term (A) and the long-term (B) studies.

Anesthesia and Surgical Preparation
One week after we initiated the high-cholesterol diet, the rabbits were anesthetized with a mixture of ketamine (5 mg/kg) and xylazine (35 mg/kg). The right carotid artery was exposed and carefully incised and a 5F sheath was inserted under fluoroscopic control into the descending aorta. An angioplasty balloon (3 mm; Advanced Cardiovascular Systems) was advanced into either iliac artery and inflated distal to the deep femoral artery six times at 8 atm with 30-second intervals between each inflation. Subsequently, the same procedure was repeated in the contralateral iliac artery. After 4 additional weeks of diet, the animals were anesthetized and the left carotid artery was cannulated for catheterization and local drug delivery.

Local Drug Delivery
A local drug-delivery balloon (3 mm; Dispatch, Scimed) was advanced to the left or right iliac artery and placed at the same position as the previous balloon-injury site. The proximal end of the delivery catheter was placed at the internal iliac branch under fluoroscopic control for landmark reference. The balloon was inflated to 6 atm and L-arginine (800 mg/5 mL) or saline was infused for 15 minutes at a rate of 0.2 mL/min (total volume, 3 mL). Subsequently, this procedure was repeated in the contralateral iliac artery. The iliac artery to receive L-arginine treatment was determined randomly. An intravenous bolus injection of cefazolin was given for prevention of infections.

Assessment of Endothelium-Dependent Vasomotion by Quantitative Angiography
After local administration of L-arginine or saline, a control angiogram was obtained. Subsequently, two infusions containing acetylcholine (10^-5 and 10^-6 mol/L) were administered at a rate of 0.8 mL/min for 3 minutes through a Swan-Ganz catheter (1.35 mm in diameter) placed above the iliac bifurcation. Immediately after each infusion, an angiogram of the iliac arteries was performed. All angiograms were measured blindly by two investigators with the use of an electronic caliper system. The diameter was measured at three predetermined sites along the area of drug delivery at baseline and after each dose of acetylcholine, before and after the local drug delivery. The vessel diameter was also measured at a reference site distal to the infusion segment to determine if there were any effects of L-arginine distal to the site of local delivery. The percent variation in diameter compared with baseline was calculated for each dose and expressed as mean±SE.

Harvesting of Tissue
Animals were killed 30 to 60 minutes after the local delivery of L-arginine, and the iliac arteries were carefully freed from adjacent tissue. Care was taken to harvest the exact portion of the artery where the local delivery was carried out by matching the anatomy with the respective fluoroscopic picture. To verify the amount of cell damage induced by the local drug-delivery balloon, electron microscopy of the segment exposed to the local delivery balloon was performed in three rabbits.

Measurements of NOs
The harvested iliac artery rings were placed in cold physiological solution. The vessel was opened longitudinally and incubated in 2 mL of Hanks' buffered saline solution (Irvine Scientific) containing calcium ionophore (1 µmol/L; A23187, Sigma Chemical Co) and L-arginine (1 mmol/L; Sigma) at 37°C.

At selected time points (0, 30, 60, and 120 minutes), samples of the medium were collected for measurement of NOx, as previously described.³⁰ After incubation, the segment was weighed and NOx was measured with a commercially available chemiluminescence apparatus (model 2108, Dasibi Environmental Corp). Samples (100 µL) were injected into a reduction chamber containing boiling acidic vanadium-chloride III. In the reduction chamber, NO₂- and NO₃- are reduced to NOx, which is then detected by chemiluminescence after reaction with ozone. Signals from the detector were analyzed by a computerized integrator and recorded as areas under the curve. Standard curves for NO₂/NO₃ were linear over the range of 50 pmol to 10 nmol.³⁰

Long-term Study (n=14)
This study was performed to determine if a single intramural administration of L-arginine could induce a persistent augmentation of NO activity and inhibit myointimal hyperplasia and/or macrophage accumulation after vascular injury (see Fig 1B).

One week after initiation of the hypercholesterolemic diet, balloon injury of the iliac arteries was performed under anesthesia as previously described. Immediately thereafter, L-arginine was administered into the wall of the right or left iliac artery via the local drug-delivery system as described. Saline was administered with the use of the same catheter system to the contralateral iliac artery. The dose of L-arginine and the infusion rate were identical to those used in the short-term study. Four weeks after balloon injury and local drug delivery, endothelium-dependent vasomotion was assessed angiographically. At 2 and 4 weeks after balloon injury and local drug delivery, vessels were harvested for histomorphometric measurements and immunohistochemistry.

Morphometric Analysis (Intima/Media Ratio)
The harvested vessels were fixed in 10% buffered formalin and then embedded in paraffin. The embedded vessels were sectioned into 5-µm-thick slices and stained with hematoxylin and eosin for light microscopy and histomorphometry. Measurements of intimal and medial cross-sectional areas were made by experienced observers blinded to the treatment group. Histological cross sections were scanned with x4 magnification and digitized with the use of the Image Analyst program (Automatix). The following borders were highlighted with a trackball: EEL, IEL, and lumen/intima border. Cross-sectional areas of the respective vessel wall layers were then calculated, and an intima (IEL-lumen)/media (EEL-IEL) ratio was calculated. The media was defined as the area between the EEL and the IEL, and the intima was defined as the vessel layer between the IEL and the intima/lumen border. Three cross sections were measured for each vessel segment affected by balloon inflation, and a mean change in diameter was calculated.

Immunohistochemistry
Immunohistochemical analysis for rabbit macrophages was performed on tissue fixed in formaldehyde and embedded in paraffin as described above. Monoclonal antibody specific for rabbit macrophage (RAM 11, Dako Corp) was used to identify macrophages. Sections were incubated with the primary antibody for 1 hour at room temperature, anti-rabbit IgG secondary antibody (biotin conjugate) for 30 minutes, and avidin peroxidase for 20 minutes. Peroxidase was then visualized with chromagen (Zymed Laboratories Inc). Three respective cross sections were immunostained for each vessel segment treated with either L-arginine or saline. Macrophage staining was assessed by two experienced observers using light microscopy. Areas of the vessel defined as media and intima and the percent of the vessel stained for macrophages were determined. The following grading was used to establish differences in the quantity of positively stained cells in the intimal area: grade I, 0 to 10%; grade II, 11% to 20%; grade III, 21% to 30%; grade IV, 31% to 50%; and grade V, >50% of intimal area covered.

NO Measurements
In four rabbits, tissue was harvested 1 week after the local delivery of L-arginine for measurements of NOx levels. Chemiluminescence measurements were performed as described above.

Data Analysis
Data are expressed as mean±SE. Changes in vasoreactivity in response to acetylcholine were expressed as percentage variations in diameter compared with baseline. The mean change of all arteries in each treatment group (L-arginine or saline) was used for comparison. A paired t test was performed to compare values between the two treatment groups for each dose of acetylcholine before and after either L-arginine or saline. Additionally, a two-factor ANOVA was performed to verify the difference within each treatment group and between the groups. Significant difference was assumed at a value of P.05. Differences between NOx levels were identified by use of Student's t test with Bonferroni correction for multiple comparisons.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Short-term Study
Vasoreactivity
Fig 2A shows the response of vessel segments to acetylcholine before and after the local delivery of L-arginine or saline. Baseline vessel diameters were identical before and after local drug delivery in both iliac arteries. There was little change in vessel diameter in response to acetylcholine before local drug delivery. After local delivery of L-arginine but not saline, endothelium-dependent vasodilatation was restored. By contrast, after local delivery of the vehicle, no vasodilatation was observed (Fig 2A). The effect of L-arginine was localized to the segment that was exposed to intramural delivery. No significant change in vessel diameter was seen at the site distal to the local delivery of L-arginine (Fig 2B). In three rabbits, electron microscopy revealed that 40% of endothelial cells were damaged by the inflation of the local drug-delivery balloon.

View larger version (21K): [in this window] [in a new window]   Figure 2. Endothelium-dependent vasomotion before and after the local delivery of L-arginine. The y axis shows percent constriction or dilatation, and the x axis shows the time course of the experiment. A, Before L-arginine, acetylcholine (Ach) did not induce vasodilation. After the local delivery of L-arginine but not saline, treated segments vasodilated in response to Ach (*P<.001). B, The vessel segments distal to the delivery site manifested little response to Ach and were not affected by L-arginine delivery to proximal segments. Ach 10-6 and Ach 10-5 indicate acetylcholine 10-6 and 10-5 mol/L, respectively.

NO Levels
Fig 3 shows the results of NOx measurements in vessel segments harvested 30 to 60 minutes after local drug delivery. Vessel segments treated with L-arginine exhibited a significant increase in NOx levels (P<.03) compared with control vessels.

View larger version (28K): [in this window] [in a new window]   Figure 3. Maximum NO production 1 hour and 1 week after local drug delivery. Vessels treated with L-arginine (hatched bars) showed significantly higher production of NO compared with vessels treated with vehicle (solid bars; *P<.04, **P<.01). Absolute values 1 week after the delivery were considerably higher than those obtained 1 hour after the delivery of L-arginine.

Long-term Study
Vasoreactivity
Vasomotion studies were performed 4 weeks after the local drug delivery. Iliac arteries treated with vehicle tended to display vasoconstriction in response to acetylcholine, whereas those treated with L-arginine tended to display vasodilation, although the observed differences did not reach statistical significance (acetylcholine 10^-6 mol/L, 2.2±1.3% versus -4.2±3.8%; acetylcholine 10^-5 mol/L, 7.2±1.0% versus -4.0±7.5%, L-arginine versus vehicle, respectively).

Intima/Media Ratio
Two weeks after the delivery of L-arginine, the intima/media ratio was significantly reduced in segments treated with L-arginine compared with control segments (0.72±0.05 versus 1.15±0.14; P.04). This phenomenon was even more apparent 4 weeks after local drug delivery (0.5±0.15 versus 1.40±0.17; P.01). Fig 4 shows representative cross sections of L-arginine– and vehicle-treated segments.

View larger version (84K): [in this window] [in a new window]   Figure 4. Low-power (x4) microphotographs of iliac arteries of hypercholesterolemic rabbits 4 weeks after balloon-catheter injury and local drug delivery. Intimal thickening is markedly reduced in the vessel segment treated with L-arginine (left) compared with that treated with vehicle (right).

Immunohistochemistry
Fig 5 shows the percentage of the intimal lesion surface area that stained positively for macrophages. Only 0% to 10% of the intimal area was infiltrated by positively stained cells in the L-arginine–treated segments. By contrast, in vessel segments treated with vehicle, the intimal area infiltrated by macrophages was markedly higher, in some segments exceeding 50%.

View larger version (21K): [in this window] [in a new window]   Figure 5. Histograms illustrating the percentage of intimal lesion occupied by macrophages. In vessel segments treated with L-arginine (hatched bars), macrophage accumulation did not exceed 20% of the intimal area. By contrast, in vehicle-treated segments (solid bars), macrophages occupied up to 70% of the intimal area in some cases.

NO Levels
In vascular segments from four rabbits, NO production was measured 1 week after L-arginine delivery. NO production ex vivo was significantly higher in those segments treated with L-arginine than in segments exposed to vehicle (Fig 3).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study reveals for the first time that intramural administration of L-arginine by use of a local drug-delivery catheter affects vascular function and structure. In the presence of endothelial injury, local administration of L-arginine increases production of NO and restores endothelium-dependent vasodilatation. After balloon injury, a single intramural dose of L-arginine enhanced NO production 1 week after treatment. The persistent enhancement of NO production produced by the single intramural administration of L-arginine reduced intimal lesion formation and suppressed macrophage accumulation in the intima 2 and 4 weeks after treatment.

This study shows a striking extended biological effect of a single dose of L-arginine. The data suggest that a single exposure to a high tissue concentration of the NO precursor L-arginine can potentially enhance production of NO and hence trigger further effects directly related to this phenomenon.

We and others^{20 21 22 23 24 25} have previously demonstrated that intravenous administration of L-arginine immediately restores endothelium-dependent vasodilator function in animals and humans and in patients with coronary artery disease. In hypercholesterolemic rabbits, long-term oral administration of L-arginine enhances NO synthesis and release as demonstrated by bioassay and chemiluminescence.^{20 21 30} The enhanced synthesis of NO is associated with reduced endothelial adhesiveness for monocytes and inhibition of intimal monocyte accumulation in the vessel wall.^{26 27 28 29 30} By contrast, long-term administration of NO synthase antagonists augments endothelial adhesiveness for monocytes and accelerates atherogenesis.^{30 31 32}

Within minutes of exposure to NO donors or to enhanced generation of endogenous NO, endothelial adhesiveness for monocytes is inhibited.³³ This immediate effect may be due to cGMP-dependent alterations in signaling of adhesion pathways. With a longer time course (hours), NO inhibits the expression of endothelial adhesion molecules and chemokines involved in monocyte adhesion.^{34 35 36} We have found that exposure of cultured endothelial cells to oxidized lipoprotein or tumor necrosis factor- induces endothelial generation of superoxide anion, activates nuclear factor-B, increases the expression of vascular cell adhesion molecule, and enhances adhesiveness for monocytes.³⁷ Prior exposure to fluid flow (to enhance NO generation) suppresses all of these effects of oxidized lipoprotein or cytokines.³⁷ Antagonism of NO synthase abrogates the effect of fluid flow whereas NO donors mimic these effects. These findings are consistent with previous observations that exogenous NO donors inhibit the endothelial expression of chemokines and adhesion molecules involved in monocyte binding by suppressing a nuclear factor-B–mediated transcriptional pathway.^{35 36} The present study extends these observations by demonstrating in vivo that local enhancement of NO activity reduces monocyte accumulation in the vessel wall. NO has also been shown to suppress the proliferation of vascular smooth muscle, which is in part mediated by a cGMP-dependent mechanism.¹² This finding may explain the observation that long-term administration of L-arginine inhibits myointimal hyperplasia after balloon injury.^{28 29} Because the endothelium is removed at the time of the intervention and is not fully regenerated at the site of injury for several weeks in this animal model, it is not likely that endothelium-derived NO is responsible for the effect of intramural L-arginine on myointimal hyperplasia. It is more likely that the activity of inducible NO synthase expressed by vascular smooth muscle cells after injury is inhibiting their proliferation. We have previously directly tested the hypothesis that generation of NO by vascular smooth muscle cells in vivo can inhibit their migration and/or proliferation. After balloon-catheter injury, transfection of vascular smooth muscle cells with a plasmid construct containing NO synthase (but not the control vector) enhanced NO generation locally and inhibited myointimal hyperplasia.³⁸

The present study is consistent with these earlier observations but extends them in several important ways. We show for the first time that a single administration of a drug by use of a local drug-delivery catheter enhances NO activity and reduces intimal lesion formation. Although previous studies have shown that drugs can be delivered into the vessel wall, no previous studies have found that a single delivery of a drug through a commercially available catheter can inhibit lesion formation or enhance vessel function. Other investigators have used different local delivery strategies with variable results. Simons et al³⁹ inhibited neointimal formation in the rat carotid artery after balloon injury by locally administering antisense oligonucleotides directed against c-myc. This local delivery approach required surgical exposure of the carotid artery to permit periadventitial application of a pluronic gel carrying the oligonucleotides. This finding has been confirmed by most investigators using a similar approach to (1) locally transfect the vessel wall with antisense oligonucleotides to suppress expression of c-myc or c-myb^{40 41} or (2) locally transfect the vessel wall with decoy oligonucleotides directed against transcriptional proteins required for cell-cycle activation.⁴² Locally delivered dexamethasone reduces neointimal formation in the rat carotid,⁴³ whereas periadventitial administration of heparin has no greater effect than intravenous infusions.⁴⁴ In all of these previous studies, administration of the agent required exposure of the selected vessel with periadventitial application of the agent or arteriotomy and direct instillation of the agent into a ligated artery. These approaches are not practical in most clinical situations. The optimal drug-delivery system would permit intravascular administration with minimal vascular injury and could be used adjunctly during other intravascular procedures.

Attempts to address this issue have resulted in the development of several technologies, such as the porous and the iontophoretic balloon catheters. Successful delivery has been achieved, but varying transit times of the drug in the vessel wall occur. The porous balloon achieves delivery of drug that can persist for 3 hours (by use of the iontophoretic balloon) and up to 1 week (by use of the porous balloon) in the vessel wall.^{45 46 47}

We chose the strategy of a prolonged intramural administration of a small volume (3 mL) of drug under pressure. Such a volume is clearly larger than the intramural space, and therefore an unpredictable amount of the delivered drug will flow downstream, since engagement of the delivery balloon with the vessel wall can never be hermetic. As a result, there will be a small quantity of the drug that leaks into the systemic circulation. In our study, this dose of L-arginine was not sufficient to have any effect just distal to the delivery site and did not improve endothelium-dependent vasomotion of the contralateral vessel.

Summary
Intramural administration of L-arginine with the use of a drug-delivery catheter enhances vascular NO generation and inhibits myointimal hyperplasia and macrophage accumulation after balloon-catheter injury. Intravascular administration of L-arginine (or other agents to enhance vascular NO generation) at the time of balloon angioplasty may represent a new therapeutic strategy to prevent restenosis.

	Selected Abbreviations and Acronyms

 

EEL = external elastic lamina IEL = internal elastic lamina NO = nitric oxide NOx = nitrogen oxide

Received July 29, 1996; revision received October 23, 1996; accepted November 17, 1996.

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