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(Circulation Research. 1996;78:337-342.)
© 1996 American Heart Association, Inc.

Articles

Chronic Inhalation of Nitric Oxide Inhibits Neointimal Formation After Balloon-Induced Arterial Injury

Joon S. Lee, Christophe Adrie, Howard J. Jacob, Jesse D. Roberts, Jr, Warren M. Zapol, Kenneth D. Bloch

From the Cardiovascular Research Center and Cardiac Unit of the General Medical Service and Department of Anaesthesia at Massachusetts General Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Kenneth D. Bloch, MD, Cardiovascular Research Center, CNY-4, Massachusetts General Hospital, 149 13th St, Charlestown, MA 02129. E-mail blochk@helix.mgh.harvard.edu.

	Abstract

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Abstract Systemic and local intravascular NO administration inhibits neointimal formation after vascular injury in animal models. NO appears to attenuate smooth muscle proliferation both directly and indirectly by preventing the release of growth factors. Inhalation of low concentrations of NO dilates pulmonary vascular smooth muscle but does not cause systemic vasodilatation. Recently, NO inhalation was found to inhibit platelet function in vivo. We studied the effects of NO inhalation on neointimal formation after balloon-induced injury of the adult rat carotid artery. Beginning 60 minutes before carotid injury, rats breathed either air with 0 or 80 ppm NO for 14 days. Rats were killed, carotid arteries were fixed and paraffin-embedded, and neointimal formation was measured by analyzing the ratio of intimal to medial areas (I/M ratio) in carotid artery cross sections. Intimal hyperplasia was evident in both groups of animals, but I/M ratios were 43% less in animals breathing 80 ppm NO for 2 weeks than in animals breathing air alone (0.78±0.12 and 1.37±0.11 [mean±SE], respectively; P<.02). Similarly, 1 week after carotid injury, neointimal formation was less in rats breathing 80 ppm NO than in rats breathing air alone (I/M ratio, 0.39±0.11 versus 0.76±0.06; P<.02). Breathing 20 ppm NO for 2 weeks or 80 ppm NO for 1 week followed by air alone for 1 week did not attenuate neointimal formation measured at 14 days. In anesthetized rats breathing 80 ppm NO or air alone for 1 hour, neither systemic blood pressure nor bleeding time differed. These observations demonstrate that inhaling 80 ppm NO inhibits neointimal formation after balloon-induced carotid artery injury in rats. NO inhalation may represent a safe and novel method of preventing restenosis after percutaneous angioplasty.

Key Words: nitric oxide • neointimal formation • angioplasty • inhalation therapy

	Introduction

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Percutaneous angioplasty is used to treat occlusive arterial disease of both the coronary and the peripheral vascular systems. Unfortunately, although initially successful in >95% of cases, in 30% to 50% of patients, a gradual renarrowing process, which is commonly termed "restenosis," occurs within 6 months.¹ This process is characterized by the migration and proliferation of smooth muscle cells in the arterial intima.^{1 2}

Success in attenuating neointimal hyperplasia in animal models by delivering NO to the area of arterial injury has been reported by several investigators. NO delivery was achieved by intravenous administration of an NO donor compound,³ local administration of a gene encoding NO synthase, the enzyme responsible for synthesis of NO from L-arginine,⁴ and stimulation of endogenous NO production by systemic administration of L-arginine.^{5 6 7} NO appears to attenuate neointimal formation directly by decreasing smooth muscle cell proliferation^{8 9 10} and indirectly by inhibiting platelet and leukocyte functions and by stimulating endothelial cell growth.^{11 12 13}

Recently, a novel method of NO delivery was developed: inhalation of low concentrations of NO gas. Inhaled NO readily diffuses into the pulmonary vasculature but, upon reaching the circulation, is rapidly inactivated by hemoglobin.^{14 15} Inhalation of low concentrations of NO gas selectively dilates the pulmonary vessels without reducing systemic blood pressure, inducing pulmonary injury, or causing methemoglobinemia.^{16 17 18 19 20 21} NO inhalation does appear to have systemic effects: Hogman and colleagues^{22 23} reported a 40% prolongation of the bleeding time in rabbits breathing 30 ppm NO. Inhalation of very high concentrations of NO (300 ppm) prolonged the bleeding time 63% but was associated with mild systemic hypotension. Moreover, in a canine model, we recently observed that the inhalation of 20 and 80 ppm NO decreased platelet-mediated coronary artery reocclusion after thrombolysis without causing systemic hypotension or methemoglobinemia.²⁴

In the present study, the ability of inhaled NO to attenuate neointimal formation after balloon-induced carotid artery injury was studied in rats. Continuous inhalation of 80 ppm NO significantly decreased the neointimal formation in response to vascular injury.

	Materials and Methods

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Arterial Injury
Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) underwent balloon-induced injury of the common carotid artery using methods previously described to induce consistent neointimal hyperplasia.²⁵ Rats weighing 300 to 350 g were anesthetized by intraperitoneal injection of ketamine (60 to 80 mg/kg) and acepromazine (0.1 mg/kg). A longitudinal midline cervical incision was made, and the left carotid artery was isolated. After dissection of the internal and external carotid bifurcation, the distal external carotid segment was ligated with a 4-0 silk suture. A small arteriotomy was made in the external carotid, and a 2F Fogarty balloon catheter (Baxter Edwards LIS) was inserted through the arteriotomy and advanced 2 cm below the carotid bifurcation. The balloon was distended with saline to 2 atm using an ACS Inflator Plus (Advanced Cardiovascular Systems Inc) and was gently withdrawn to the level of the bifurcation. The balloon was deflated, and the balloon-induced injury was repeated for a total of three times. The balloon catheter was then withdrawn, and after allowing backbleeding through the arteriotomy site to eliminate any potential thrombus or air bubbles, the external carotid was ligated proximal to the arteriotomy site using 4-0 silk sutures. After visual inspection to ensure adequate pulsation of the common carotid artery, the surgical incision was closed, and the rats were allowed to recover from anesthesia.

Chronic NO Inhalation
Rats breathed NO in specially prepared 40-L acrylic inhalation chambers.²⁶ The gas mixtures were blended using separately regulated and calibrated flowmeters for oxygen, pressurized air, and NO stock gases (800 and 10 000 ppm, Airco). The oxygen concentration in the effluent gas from the chamber was analyzed periodically throughout the experimental period to ensure an FIO₂ of 0.21 using a polarographic electrode (Oxygen Monitor, Hudson Ventronics Division). Concentrations of NO, as well as nitrogen dioxide and nitrogen with higher oxidation states (NO_x), in the effluent gas were measured with an NO/NO_x chemiluminescence analyzer²⁷ (model 14A, Thermo Environmental Instruments Inc). Soda lime (J.T. Baker Chemical Co), which was maintained in the chambers to reduce the levels of NO_x, was replaced twice a week. NO concentration was maintained at 20 or 80 ppm, depending on the experiment. Serial measurements revealed that NO_x levels were 6% of the NO concentration. The gases exiting the exposure chambers, as well as those discharging from the chemiluminescence instrument, were scavenged with a Venturi trap maintained at negative atmospheric pressure by the laboratory's central vacuum system.

Control animals were maintained in filtered cages in the same room as the NO-treated animals. NO exposure was begun 60 minutes before surgery. During the surgical procedure, the NO-treated group was taken out of the chamber and exposed to NO by a modified face mask fed from the inflow gas tubing of the chambers. Analysis of the gas in the face mask revealed NO levels of 50 to 80 ppm during the 80-ppm experiments and 10 to 20 ppm during the 20-ppm studies. The total duration of time out of the chamber and under the face mask ranged from 20 to 40 minutes.

Rats were given free access to water and rat chow throughout the study. Fifteen rats breathing air alone, 8 rats breathing 20 ppm NO, and 7 rats breathing 80 ppm NO were weighed before carotid injury and at the time of death 14 days later.

Morphometric Analysis
Rats were euthanized by an intraperitoneal injection of sodium pentobarbital. A 16-gauge catheter was introduced into the ascending aorta via the left ventricular apex. The descending thoracic aorta was ligated, and the right atrium was vented. The vasculature was then perfused at a pressure of 100 mm Hg with 100 mL of normal saline, followed by 50 mL of 2% paraformaldehyde in PBS. After in vivo fixation for 15 to 30 minutes, both common carotid arteries were excised and underwent additional fixation overnight in PBS containing 4% paraformaldehyde. The tissue was dehydrated using sequentially increasing concentrations of ethanol followed by xylene and embedded in paraffin. Cross sections (6 µm) were cut and stained with hematoxylin and eosin and/or elastin stain for analysis. For each animal, a single section 7 to 8 mm proximal to the carotid bifurcation was photographed, and the image was digitized at 2000 dots per inch using a Kodak RF 2035 Plus Film Scanner (Eastman Kodak Co). The carotid artery intimal and medial areas were calculated using an image analysis computer program (NIH Image). Section preparation, photography, and morphometric analysis were performed by an investigator blinded to the experimental group.

Bleeding Time and Blood Pressure Measurements
Twenty rats weighing 350 to 450 g were anesthetized as described above. For each rat, the right common carotid artery was cannulated with a polyethylene catheter (model PE-50, Intramedic, Becton Dickinson), and a tracheostomy was performed. After paralysis with 0.2 mg/kg pancuronium (Gensia Pharmaceuticals, Inc), each animal was ventilated at 50 breaths per minute and a tidal volume of 5 mL/kg with a rodent ventilator (model 683, Harvard Apparatus). FIO₂ was kept constant at 0.21. Carotid arterial blood pressure was continuously measured with calibrated pressure transducers (Cobe Laboratories) and recorded (model 7754, Hewlett Packard). Bleeding times were performed at room temperature with the tip of the tail 10 cm below the platform upon which the animal was placed. Bleeding was induced by transection of the tail 5 mm from the tip. The tails were gently blotted with filter paper, and the time in seconds to cessation of bleeding was noted by an investigator blinded to the inhaled NO concentration.

After ventilation with air for a 20-minute stabilization period, rats were ventilated with either 0 (n=10) or 80 (n=10) ppm NO for 60 minutes. Bleeding times were performed at the end of this exposure period. To ensure consistent oxygenation and ventilation, heparinized blood samples were obtained from the carotid artery during the stabilization period and during the 60-minute exposure period, and blood gas tension and pH were measured on a blood gas analyzer (model 238, Ciba Corning Diagnostics Ltd).

Statistical Analysis
All data are presented as mean±SE. Intimal and medial areas and ratios of intimal to medial areas (I/M ratios) of the four groups of rats studied 14 days after carotid injury were compared by a factorial model of ANOVA. When significant differences were detected, Scheffé's analysis was used post hoc to compare groups. Intimal and medial areas and I/M ratios of the two groups of rats studied 7 days after carotid injury were compared using an unpaired Student's t test. Significance was declared at P<.05.

	Results

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A total of 82 rats underwent the carotid artery injury procedure. There were no postprocedure deaths. All rats, regardless of their experimental group, exhibited normal grooming behavior and activity levels throughout the study. Other than occasional ptosis on the side of the carotid injury, no rats exhibited a gross neurological deficit. No significant differences were observed in weight gained over the 2-week experimental period in rats breathing 0, 20, or 80 ppm NO (data not shown).

In the first series of experiments, the rats undergoing balloon-induced injury to the carotid artery breathed either air (n=35) or 80 ppm NO in air (n=20) throughout the duration of the study. The animals were killed 14 days after injury for morphometric analysis of the injured carotid artery. Upon light microscopic examination, three rats breathing air alone and one breathing NO were found to have thrombotic occlusion of the lumen of the injured carotid artery. These four animals were excluded from further analysis. Vessels harvested from both groups exhibited loss of endothelium and the development of neointimal hyperplasia as described previously by other investigators.²⁵ Examination by light microscopy did not reveal a qualitative difference in the cellular morphology of the neointima between animals breathing NO and those that did not (Figure). Quantitative analysis revealed that the intimal area was 45% less in rats breathing 80 ppm NO than in rats breathing air alone (P<.04), whereas medial areas did not differ between the two groups (Table). The I/M ratio was 43% less in animals inhaling 80 ppm NO for 14 days than in animals breathing air (P<.02).

View larger version (0K): [in this window] [in a new window]   Figure 1. Cross sections of balloon-injured rat carotid arteries (original magnification x100). Sections of carotid arteries are shown 14 days after injury from rats exposed to air alone (top) and exposed to air with 80 ppm NO (bottom). The sections shown represent carotid arteries with the median intimal-to-medial ratios for the two groups.

View this table: [in this window] [in a new window]   Table 1. Morphometric Analysis of Injured Rat Carotid Arteries

The effect of breathing NO on neointimal formation was also examined 1 week after carotid injury. Neointimal formation was less in carotid arteries 1 week after injury than 2 weeks after injury. One week after carotid injury, inhaling 80 ppm NO decreased neointimal formation by 55% (P<.01) but did not alter the medial area. The I/M ratio in injured carotid arteries from rats breathing 80 ppm was 49% less than that in arteries from rats breathing air (P<.02).

Two additional groups of animals were studied. To investigate the effect of breathing a lower concentration of NO on the neointimal response, 8 rats subjected to carotid injury were exposed to 20 ppm NO for 2 weeks and then killed. In these rats, the carotid artery I/M ratio was 0.92±0.23 and did not differ from that observed in rats breathing air alone. To determine whether shorter exposures to NO were sufficient to attenuate neointimal formation, 7 rats subjected to carotid artery injury breathed air with 80 ppm NO for 1 week, followed by breathing air alone for a second week. In these rats, the I/M ratio was 1.06±0.24 and was not different from that observed in rats breathing air for 2 weeks.

To investigate the effect of NO inhalation on systemic platelet function, tail transection bleeding times were measured in anesthetized rats breathing NO for 1 hour. Bleeding time did not differ between 10 rats breathing air (213±20 seconds) and 10 rats breathing 80 ppm NO (246±18 seconds). Systemic blood pressures were also measured in these animals. Mean arterial blood pressures did not differ significantly in rats breathing air (69±3 mm Hg) or 80 ppm NO (78±5 mm Hg).

	Discussion

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In the present study, chronic inhalation of low concentrations of NO gas was found to inhibit neointimal formation after balloon-induced vascular injury. Breathing 80 ppm NO decreased neointimal formation 1 and 2 weeks after injury (Table). To achieve this inhibition, the rats needed to continuously breathe NO: inhaling NO for the first 7 days after injury followed by 7 days of air breathing did not inhibit neointimal formation at 14 days. Breathing a lower concentration of inhaled NO (20 ppm) for 2 weeks did not attenuate the neointimal response to injury.

Ventilation of anesthetized rats with 80 ppm NO did not alter systemic blood pressure. Although exposures of unanesthetized rats might have provided different results, studies in awake sheep revealed that breathing 80 ppm NO did not alter systemic blood pressure.¹⁶

Although inhibition of neointimal formation by local or systemic delivery of NO donor compounds has been observed previously,^{3 5 6 7 28} this is the first report of producing such an effect using the novel respiratory therapy, NO inhalation. The magnitude of inhibition achieved by inhaling 80 ppm NO was comparable to that reported for the administration of L-arginine.^{5 6 7} Our findings were also similar to those of Guo et al,³ who observed that intravenous administration of the NO donor compound SPM-5185 decreased neointimal formation in rat carotid arteries 7 days after desiccation-induced intimal injury and did not cause systemic hypotension.

Several mechanisms potentially account for the inhibition of neointimal formation by NO inhalation. Many investigators have postulated that the effect of NO on neointimal hyperplasia is mediated by its ability to directly inhibit smooth muscle cell proliferation at the site of injury. This is supported by in vitro experiments showing that NO donor compounds suppress the proliferation of isolated vascular smooth muscle cells^{3 8 9 29} ; however, recent evidence suggests that under certain conditions NO may also stimulate smooth muscle cell mitogenesis.³⁰

Because NO is almost immediately inactivated in the presence of hemoglobin, NO has an extremely short half-life in blood of 111 to 130 milliseconds.²⁹ Using chemiluminescence, Rich et al¹⁵ measured the concentration of NO in the effluent of isolated perfused rat lungs ventilated with 1000 ppm NO; they observed that if the lungs were perfused with whole blood, NO was undetectable in the effluent. The rapid inactivation of NO by hemoglobin accounts for the ability of inhaled NO to produce local pulmonary vasodilatation without reducing the systemic vascular resistance or systemic arterial pressure in animal models and in humans.^{16 17 18 19 20 21} Therefore, it is unlikely that NO absorbed into the pulmonary circulation decreases neointimal formation at the site of carotid injury via a direct effect.

Under certain conditions, NO can react with thiol-containing molecules, which then have NO-like vasodilatory and platelet inhibitory properties.³¹ NO bound to thiols has a longer functional half-life in blood than does free NO. During NO inhalation, S-nitrosothiols may be produced in the pulmonary vasculature or formed in the airways³² and then absorbed into the circulation. These S-nitrosothiols could travel to the site of arterial injury and produce the same effect on neointimal formation as NO donor compounds. Rimar and Gillis¹⁴ measured the vasodilator activity present in the effluent of isolated perfused rabbit lungs ventilated with 150 ppm NO; they observed that even small amounts of blood in the perfusate blocked the ability of the effluent to vasodilate aortic rings. These results suggest that circulating S-nitrosothiols formed during NO inhalation possess minimal vasodilator properties or are present in very low concentrations and are unlikely to directly inhibit neointimal formation.

Another mechanism potentially accounting for the effect of inhaled NO on neointimal formation may be that exposure of one or more circulating blood elements to NO, during transit through the pulmonary circulation, modulates their function at the site of vascular injury, inhibiting neointimal development. Through this mechanism, inhaled NO would be able to exert a systemic vascular effect despite being anatomically limited to the pulmonary vasculature. Although the design of the present study does not directly reveal which circulating blood element mediates the effect of inhaled NO, candidates include platelets and leukocytes.

Many investigators have demonstrated the platelet-inhibitory effects of NO and NO donor compounds.^{13 33 34} In vitro, NO inhibits platelet adhesion,^{35 36} activation,³⁷ aggregation,^{35 38} and mitogen release.³⁹ In vivo, systemic administration of NO donor compounds to animals and humans prolongs the bleeding time^{40 41} and inhibits platelet adherence to atherosclerotic plaques,⁴² as well as to balloon-injured arteries.^{3 43 44} The importance of platelets for neointimal formation after vascular injury is well established. Friedman et al⁴⁵ demonstrated prevention of neointimal formation after balloon-induced injury in rabbits by inducing thrombocytopenia using antiplatelet antisera. In the injured rat carotid artery model, Fingerle et al⁴⁶ observed that induction of thrombocytopenia for 48 to 96 hours attenuated neointimal formation measured 4 and 7 days after injury.

In contrast to the observations of Hogman and colleagues,^{22 23} who reported a modest prolongation of the bleeding time in humans and animals during NO inhalation, we did not observe a prolongation of the tail-transection bleeding time in rats breathing 80 ppm NO for 1 hour. The tail-transection bleeding times that we measured, which reflect both platelet and coagulation function, were similar to those reported by de Gaetano and colleagues.^{47 48} It is possible that shorter or longer durations of exposure to NO might have prolonged the bleeding time. The failure of inhaled NO to prolong the bleeding time does not exclude the possibility that inhaled NO attenuated neointimal formation by altering systemic platelet function. Samama et al⁴⁹ recently reported that inhalation of up to 100 ppm NO by six patients with adult respiratory distress syndrome did not prolong the bleeding time but did decrease ex vivo platelet aggregation. We observed that inhalation of 20 and 80 ppm NO increased vascular patency in a canine model of platelet-mediated coronary artery thrombosis after thrombolysis²⁴ but did not prolong the bleeding time measured on the ventral aspect of the tongue and on the shaved forepaw (authors' unpublished data, 1995).

Another potential cellular mediator of the effect of inhaled NO on neointimal formation is the leukocyte. It is accepted that leukocytes, especially monocytes, play an important role in the pathogenesis of primary atherosclerosis as well as restenosis.⁵⁰ In vitro, NO inhibits many leukocyte functions, including adhesion⁵¹ and chemotaxis.¹² It remains to be determined whether NO inhalation alters systemic leukocyte function or whether NO-exposed leukocytes modulate neointimal formation.

Based on these observations from a single rodent model of vascular injury, it cannot be concluded that inhaled NO will prevent restenosis after percutaneous coronary angioplasty in humans. However, several features of inhaled NO therapy suggest that it may be clinically useful. First, NO gas may be readily administered via face mask or nasal prongs. Second, no significant side effects have been reported associated with inhaling NO in the low concentrations used in clinical studies (up to 80 ppm). Third, NO inhalation in humans, as well as rats, does not cause systemic hypotension. Fourth, breathing NO causes little or no prolongation of the bleeding time in the four species studied (human, dog, rabbit, and rat), suggesting that its clinical use will be associated with a low risk of hemorrhagic complications. A potential disadvantage of inhaled NO for the prevention of neointimal formation is that continuous inhalation appears to be necessary: neointimal formation in animals breathing 80 ppm NO for 1 week followed by breathing air for 1 week did not differ from that in animals breathing air alone for 2 weeks. This latter observation is similar to the finding that transient thrombocytopenia decreased neointimal formation when measured 1 week after injury but not when measured 2 weeks after injury.⁴⁶ It is conceivable that agents, such as selective inhibitors of type V cGMP phosphodiesterase,⁵² will be used to augment and/or prolong the effect of inhaled NO or to permit intermittent administration. Alternatively, inhaled NO may be used to postpone neointimal formation, during which time other agents may be used, for example, to accelerate reendothelialization of injured vascular segments.⁵³

In summary, inhalation of 80 ppm NO inhibits the neointimal response to balloon-induced injury of the rat carotid artery. NO inhalation did not cause systemic hypotension or prolong the bleeding time. These results suggest that inhaled NO may provide a safe respiratory therapy to inhibit clinical restenosis.

	Acknowledgments

 
This study was supported by National Institutes of Health grants T32 HL-07208 (Dr Lee), HL-42397 (Drs Zapol and Roberts), and HL-45895 (Dr Bloch) and by a grant to the Cardiovascular Research Center from Bristol Myers Squibb Pharmaceuticals. We would like to thank Chris Simpson for his expert assistance and advice in the preparation of the histological sections and Jonathan Delgado for his help with the computer software.

Received September 5, 1995; accepted November 22, 1995.

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