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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:737-740.)
© 1997 American Heart Association, Inc.

Articles

Induction of Nitric Oxide Synthase in the Neointima Induced by a Periarterial Collar in Rabbits

J.F. Arthur; Z.L. Yin; H.M. Young; ; G.J. Dusting

From the Departments of Physiology and Anatomy and Cell Biology (H.M.Y.), University of Melbourne, Parkville, Victoria, Australia.

Correspondence to Prof G.J. Dusting, Department of Physiology, The University of Melbourne, Parkville, Victoria 3052, Australia. E-mail g.dusting{at}physiology.unimelb.edu.au

	Abstract

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Abstract A Silastic collar placed around the common carotid artery of rabbits causes the formation, within 7 days, of an atheroma-like neointima containing cells with the appearance of synthetic-phenotype smooth muscle cells. Using immunohistochemistry, we detected the appearance of the cytokine-inducible form of nitric oxide synthase (iNOS, or isoform II) in the neointima of rabbits that had the collar in place for 7 or 14 days. This iNOS immunofluorescence collocalized with anti–smooth muscle myosin in the intima, indicating that it is expressed in smooth muscle cells, and iNOS was also present in a few endothelial cells in collared sections. There was no evidence of iNOS expression in the arterial wall before the neointima was apparent, that is, after only 2 days with the collar. The expression of endothelial NOS (eNOS, or isoform III) immunofluorescence was confined to the endothelial cells in control sections, as it was in collared sections with neointima at 7 and 14 days. Specific immunofluorescence for neuronal NOS (nNOS, or isoform I) was not observed in any sections. Our results suggest that nitric oxide is produced by the inducible isoform of NOS in modified smooth muscle cells of the developing neointima. Activity of iNOS might deprive the endothelium of substrate for nitric oxide production and might explain the compromised endothelium-dependent vasodilatation observed both in this model of atherosclerosis and in human coronary artery disease.

Key Words: atherosclerosis • neointima • nitric oxide • nitric oxide synthase • periarterial collar

	Introduction

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The release of NO by endothelial cells plays a vital role in the regulation of blood pressure and regional blood flows. A cNOS, which is responsible for the production of NO from L-arginine, is present in the endothelium,¹ but other isoforms of NOS may also be expressed in these cells.^{2 3 4} The vascular endothelium, platelets, and the brain produce NO via constitutively expressed, calcium-dependent NOSs. nNOS and eNOS have been classified as types I and III, respectively.⁵ cNOSs are responsible for short periods of release of NO (in picomolar amounts) in response to receptor-stimulated or physical stimuli, such as rapid changes in shear stress on the endothelium.^{4 6 7}

Lipopolysaccharide endotoxins and cytokines increase the production of NO by inducing the expression of a calcium-independent form of NOS. iNOS,⁵ expressed in vascular smooth muscle cells, macrophages, and endothelial cells, unlike the constitutive enzyme, is fully active in the absence of calcium and calmodulin.⁸ The expression of iNOS develops over several hours in response to bacterial lipopolysaccharides and specific cytokines.^{9 10 11} Induction of this enzyme results in the formation of nanomolar amounts of NO.¹²

The expression of a calcium-independent iNOS activity has been reported to be elevated in the myocardium of rabbits after myocardial infarction,¹³ in macrophages and myocytes of rats during cardiac transplant rejection,¹⁴ and in human patients with dilated cardiomyopathy.¹⁵ Although there is considerable evidence that endothelium-dependent vasodilatation is impaired in atherosclerotic blood vessels,⁷ there have been reports that production of nitrogen oxides is actually increased in these vessels,^{16 17 18} suggesting increased activity of NOSs. We have been studying the formation of a neointima induced by the application of a nonconstricting Silastic collar to the common carotid artery of rabbits, which produces, within 7 days, a neointima with characteristics similar to those of human early atheroma.^{7 19 20 21} Functionally, the intima-bearing (collared) arterial segments exhibit impaired vasodilatation to the endothelium-dependent vasodilator acetylcholine,^{21 22} which acts by releasing NO from the endothelium. This impairment is evident before intimal thickening has occurred²² and persists when the neointima has developed. The aim of this study was to investigate the isoforms of NOS present in the wall of the rabbit carotid artery and to determine whether the expression of these isoforms is altered during development of the neointima.

	Methods

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Surgery
A hollow, flexible, nonocclusive Silastic collar 2 cm long and in contact circumferentially with the adventitia for a length of 3 mm at each of its ends, as described by Booth et al¹⁹ and Dusting et al,²⁰ was applied to each common carotid artery of rabbits (2.5 to 3.5 kg).

Tissue Preparation
Carotid arteries were removed from rabbits at 2 (n=2), 7 (n=5), or 14 (n=3) days after surgery and placed in ice-cold PBS (0.9% NaCl in 0.01 mol/L sodium phosphate buffer, pH 7.0) while they were cleaned of adherent connective tissue and then cut into segments. These were a control section taken at least 0.5 cm outside the collar and center sections of the collared segment divided at its center. Segments to be used in immunohistochemical processing for iNOS, nNOS, and substance P were then immersed in Zamboni's fixative (2% formaldehyde and 0.2% picric acid in 0.1 mol/L sodium phosphate buffer, pH 7.0) for 24 hours at 4°C. Segments to be used in immunohistochemical processing for eNOS were immersed in 4% paraformaldehyde for 24 hours at 4°C. After fixation, the Zamboni-fixed tissue was cleared of fixative with three 10-minute washes in DMSO, followed by three 10-minute washes in PBS. The paraformaldehyde-fixed segments were washed in three changes of PBS (3x10 minutes). After clearing, all arterial segments were placed in PBS containing 0.1% sodium azide and 30% sucrose (as a cryoprotectant) for 3 hours to allow the sucrose to penetrate the artery. The segments were then transferred to 50% OCT embedding medium (Tissue-Tek, Miles Inc) and 50% sucrose/azide solution overnight at 4°C to allow penetration of OCT into the tissue.

The following day, each segment of artery was positioned in a cryomould containing OCT and frozen in a beaker of isopentane that had been cooled in liquid nitrogen. Serial sections 10 µm thick were cut from the same arterial segment of day 2, 7, or 14 arteries onto gelatin-coated slides and air-dried for at least 60 minutes.

Fluorescence Immunohistochemistry
Slides were preincubated for 1 hour in 10% normal sheep serum or in 10% normal horse serum in a humid box at room temperature. The serum was then removed by careful blotting of the slides. The primary antibodies, a rabbit antiserum to NOS isoform II from murine RAW 264.7 macrophages (provided by Dr R. Tracey) diluted at 1:400, a rabbit polyclonal antiserum to iNOS (Transduction Laboratories) diluted at 1:100, a rabbit polyclonal antiserum to eNOS (Transduction Laboratories) diluted at 1 µg/mL, an antiserum raised against nNOS (N74²³ ) diluted at 1:200, or a mouse antibody to smooth muscle myosin (Sigma) diluted at 1:1000, were then applied, and the sections were left in a humid box at room temperature to incubate overnight. Antiserum to substance P²⁴ diluted at 1:2000 and normal rabbit serum diluted at 1:400 were also used on the sections as a negative control. The following day, sections were washed (3x10 minutes) in PBS and then incubated in the secondary antibody, a sheep or donkey anti-rabbit IgG conjugated to FITC or a rabbit anti-mouse IgG conjugated to FITC, for a further 1 hour at room temperature. After the incubation in the secondary antibody, the slides were washed as before with PBS. Chicago sky blue (0.05% in 1% DMSO) was applied to the sections for 90 seconds to reduce the autofluorescence of the elastic components of the arterial segments. The sections were then mounted under coverslips in buffered glycerol (pH 8.6) for examination under a fluorescence microscope (Axioplan, Zeiss). Photographs were taken with Kodak TMax 1000 ASA film.

	Results

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Two Days
Examination of the arterial sections under a light microscope revealed that 2-day sections exhibited no morphological evidence of lesion development, confirming previous findings.^{20 22} No specific fluorescence was detected in either control or collared sections of artery after immunohistochemical processing for iNOS, nNOS, or substance P. There was fluorescence in the endothelial layer of both control and collared sections after processing with antibodies to eNOS. Fluorescence was evident in the adventitia, but this was found to be nonspecific, because sections incubated with normal rabbit serum alone followed by FITC-labeled secondary antibody also exhibited adventitial fluorescence.

7 Days and 14 Days
A substantial neointima consisting of several cell layers was present in sections taken from the center region of carotid arteries enclosed in the collar for 7 or 14 days.

At 7 days, immunohistochemical processing using the iNOS antibody revealed intense fluorescence in the neointima (Fig 1A). There was no specific fluorescence after immunohistochemical processing for either nNOS (Fig 1B) or substance P. Immunohistochemical processing for eNOS in collared arterial sections revealed that eNOS was evident in the endothelial cells (Fig 1C). In some arteries, the endothelial cells showed signs of modest proliferation (Fig 1C). Nonspecific fluorescence was evident in the adventitia of all sections, including sections that were exposed to normal rabbit serum and secondary antisera.

View larger version (83K): [in this window] [in a new window]   Figure 1. Fluorescence micrographs demonstrating localization of different isoforms of NOS in collar and control sections taken from rabbit common carotid arteries 7 days after collar placement. Arrows indicate internal elastic lamina; i, neointima; m, media; and e, endothelium. Scale bar=50 µm. A, iNOS immunoreactivity is present in collar-induced neointima but not in media. B, After immunohistochemical processing for nNOS, no staining is observed in neointima or in media. C, Immunohistochemical processing for eNOS revealed staining of endothelial cells (e) in collared arterial segments but no significant staining of neointimal or medial smooth muscle cells. Endothelium appears to have proliferated somewhat in this section, although this is not typical. D and E, Control carotid artery processed for iNOS immunohistochemistry (D) or eNOS immunohistochemistry (E). There is no specific iNOS immunoreactivity in any part of artery wall (D), and immunoreactivity for eNOS is restricted to endothelial cells (E).

At 14 days, the localization of iNOS, eNOS, and smooth muscle myosin was examined in serial sections from the center of the collared region. Immunohistochemical processing using the iNOS antibody revealed that staining in the neointima was similar to that at 7 days, with the additional staining of a few endothelial cells (Fig 2A). Immunoreactivity to eNOS was present only in the endothelial cells (Fig 2B). Immunoreactivity to smooth muscle myosin was present in most cells of the neointima and media but not in the endothelial cell layer (Fig 2C).

View larger version (41K): [in this window] [in a new window]   Figure 2. Fluorescence micrographs demonstrating localization of iNOS (A), eNOS (B), and smooth muscle myosin (C) taken from rabbit common carotid artery 14 days after collar placement. Arrows indicate internal elastic lamina; arrowheads, endothelial cells; i, neointima; and m, media. Scale bar=25 µm. A, Immunoreactivity to iNOS is present in neointima and in some endothelial cells (arrowheads) but not in media. B, Immunoreactivity to eNOS is present only in endothelial cells. C, Immunoreactivity to smooth muscle myosin is present in neointima and media but not in endothelial cells.

Sections of control regions of artery revealed no fluorescence after immunohistochemical processing using the iNOS (Fig 1D), nNOS, or substance P antibodies, whereas there was strong fluorescence of the endothelial cells with immunohistochemical processing for eNOS (Fig 1E).

The demonstration of iNOS in the neointima is consistent with data previously obtained in organ bath studies. In arterial segments from 7-day collared rabbits, the addition of N^G-nitro-L-arginine, an inhibitor of NOS, produced large increases in resting tone in collared segments but had little effect in controls (Fig 3). This suggests that there is a higher basal release of NO in collared arterial segments than in controls.

View larger version (13K): [in this window] [in a new window]   Figure 3. a, Contractions of control and collared arterial segments in organ bath at basal tension after 30-minute incubation with N-nitro-L-arginine (NOLA). Data are mean±SEM (n=7). *P<.05, Student's paired t test. b, Representative trace showing increase in resting tension after addition of NOLA in a collared arterial segment and a control segment from same artery.

	Discussion

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The most important observation of this study was the expression of iNOS in the neointima of rabbits after 7 to 14 days of neointima development. Stimulation of cultured smooth muscle cells by cytokines produces high levels of iNOS.²⁵ Functional studies suggest that NOS is induced in balloon-injured rat carotid arteries,²⁶ because attenuated contractions induced by phenylephrine in injured arteries at 6 and 24 hours after balloon de-endothelialization could be restored by inhibitors of NOS. It has also been reported recently that de-endothelialization of rat carotid arteries induces the expression of NOS in neointimal smooth muscle cells.²⁷ In that study, mRNA for iNOS appeared in the injured vessel at 24 hours after the removal of the endothelium. We could not detect iNOS in collared arterial segments 2 days after surgery because in our hands the endothelium remains intact after placement of the collar and subsequent development of the neointima.

In the present study, double immunostaining for iNOS and smooth muscle myosin revealed that the modified smooth muscle cells that make up 98% of the neointima²⁸ are the intimal cells in which iNOS is expressed. iNOS also appeared in a few endothelial cells of the collared sections, but contractile smooth muscle cells of the media did not express iNOS. In one rabbit that did not develop a significant neointima 7 days after surgery, there was no expression of iNOS, nor was there any in collared arterial segments 2 days after collar placement, before there is morphological evidence of lesion development. Thus, iNOS appears to be expressed only in the modified smooth muscle cells (and occasional endothelial cells) of the neointima and is therefore associated with the change from contractile to synthetic phenotype.²⁹

The expression of endothelial cell NOS and release of nitrogen oxides is markedly increased in proliferating compared with quiescent cells,³⁰ so it is possible that the proliferation of modified smooth muscle cells during the formation of the neointima might result in the induction of NOS in these cells. Various cytokines, such as interleukins-1 and -2, interferon-, and tumor necrosis factor-, are powerful stimulators of iNOS gene expression, and there is abundant evidence that these are produced by cells within atheroma-like lesions, particularly in macrophages or T cells present. Consistent with this, Minor et al¹⁶ reported a markedly increased output of nitrogen oxides from atherosclerotic rabbit aorta, despite loss of NO bioactivity. This suggests that iNOS is responsible for increased production of NO in atherosclerotic lesions but that the mediator is biologically neutralized, probably because of inactivation by superoxide radicals produced in these lesions.⁷

At this time it is unclear whether the expression of iNOS in lesions is directly related to the abnormal endothelium-dependent vasodilatation observed in lesioned vessels. Certainly, from immunohistochemical evidence, eNOS was clearly expressed in collared sections at all stages of lesion development, both before and after intimal thickening. However, it is conceivable that although eNOS is present, it might have altered function in these lesions. Given that NO itself acts as a negative feedback modulator of the endothelial NOS,^{31 32 33} it is possible that excess production of NO by iNOS might compromise the activity or expression of the cNOS, which is stimulated by endothelium-dependent vasodilators. Alternatively, iNOS in neointimal cells may act as a sink for the L-arginine substrate, stealing substrate from eNOS and thus compromising moment-to-moment regulation of blood flow by the endothelium.⁷

Given the deleterious effects of excess NO production (including cytotoxicity and the possibility that it downregulates cNOS activity), it would be interesting to study the action of a specific inhibitor of iNOS in the development of atherosclerosis. Impairment of eNOS function that normally accompanies the development of these lesions creates conditions propitious for vasoconstriction and thrombosis, and specific inhibitors of iNOS might therefore help preserve eNOS function, thus alleviating the secondary consequences of atherosclerotic artery disease.

	Selected Abbreviations and Acronyms

 

cNOS = constitutive nitric oxide synthase eNOS = endothelial nitric oxide synthase(isoform III) iNOS = inducible nitric oxide synthase(isoform II) nNOS = neuronal nitric oxide synthase(isoform I) NOS = nitric oxide synthase

	Acknowledgments

 
This study was supported by grants from the NH & MRC (Australia) and National Heart Foundation of Australia. We are indebted to Dr W. Ross Tracey (Pfizer) for the gift of the iNOS antibody and to Dr Colin Anderson and Prof John Furness for the nNOS antibody. We thank Prof Furness for his advice and use of facilities.

Received July 19, 1996; accepted August 21, 1996.

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This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Arthur, J.F. Articles by Dusting, G.J. Search for Related Content PubMed PubMed Citation Articles by Arthur, J.F. Articles by Dusting, G.J.