Vasculoprotective Role of Inducible Nitric Oxide Synthase at Inflammatory Coronary Lesions Induced by Chronic Treatment With Interleukin-1β in Pigs in Vivo
Background We recently developed a porcine model in which chronic, local treatment with interleukin-1β (IL-1β) causes coronary arteriosclerotic changes and hyperconstrictive responses. Inflammatory cytokines are known to induce inducible NO synthase (iNOS) in the vascular smooth muscle. This study was designed to examine whether or not the production of NO by iNOS has a protective or deleterious effect on the coronary artery in vivo.
Methods and Results A segment of the porcine coronary artery was aseptically wrapped with cotton mesh absorbing IL-1β suspension. We inhibited both eNOS and iNOS activity by cotreatment with L-NAME (a nonspecific inhibitor of NOS) and iNOS activity alone by aminoguanidine (a selective inhibitor of iNOS). Immunostaining showed that iNOS was absent in the normal coronary artery, whereas it was highly expressed 1 day after the application of IL-1β and thereafter downregulated until 14 days. In contrast, eNOS was well maintained throughout the study period. Two weeks after the operation, hyperconstrictive responses to intracoronary serotonin and neointimal formation were noted at the IL-1β–treated site, and both responses were significantly greater at the site cotreated with either L-NAME or aminoguanidine.
Conclusions These results indicate that iNOS is transiently induced in vivo in response to local inflammation and that NO produced by iNOS exerts an inhibitory effect against the cytokine-induced proliferative/vasospastic changes of the coronary artery in vivo.
Increased coronary vasomotion or coronary artery spasm plays an important role in the pathogenesis of a wide variety of ischemic heart diseases1,2; however, the mechanism of this disorder still remains to be elucidated. We previously developed a swine model of coronary artery spasm and demonstrated the importance of the atherosclerotic changes in the pathogenesis of coronary spasm.3-5 However, the aspect of coronary atherosclerosis responsible for the occurrence of this disorder remains to be examined.
Atherosclerosis is an excessive and inflammatory/proliferative response of the vascular wall to various forms of injury.6,7 Those injuries include not only mechanical but also inflammatory and immunological types of injury.6-9 Recent studies in vitro have shown that inflammatory cytokines stimulate the proliferation of vascular smooth muscle cells, which suggests that they play an important role in the pathogenesis of atherosclerosis.6-9 We recently developed a new swine model of coronary spasm in which vasospastic responses to autacoids can be induced at the site of the coronary artery where IL-1β, a major inflammatory cytokine in atherosclerotic lesions, was applied locally and long-term.10,11 This new swine model has demonstrated the importance of the inflammatory changes of the coronary artery in the pathogenesis of coronary spasm.10,11
Conversely, a number of studies have also revealed that inflammatory cytokines, including IL-1β, induce NOS in both vascular smooth muscle cells12-14 and cardiac myocytes.14,15
A large amount of NO produced by iNOS16 is reported to be responsible in part for the sustained reduction in cardiac contraction and for the sustained, internal vasodilatation in sepsis.17,18 A small amount of NO produced by eNOS in the endothelium demonstrates various vasculoprotective actions19,20; however, the role of a large amount of NO produced by iNOS in vascular smooth muscle cells remains to be examined. In contrast, TNF-α, another major inflammatory cytokine, is reported to reduce the expression of eNOS by destabilizing the eNOS mRNA in vitro.21 However, whether this is also the case in vivo remains to be elucidated. The present study was therefore designed to determine how the vascular NOS system is altered in response to local inflammation and whether such alterations in the vascular NOS system have a protective or deleterious effect on the coronary artery in vivo.
A total of 32 male domestic pigs (Nihon Crea Inc, Tokyo; 2 to 3 months old; weight, 25 to 30 kg) were used. Twelve animals were used for immunostaining alone (protocol 1), and the other 20 were used for angiographic and histological studies (protocols 2 and 3). The animals were housed individually at a controlled room temperature. They were sedated with ketamine hydrochloride (12.5 mg/kg IM) and were then anesthetized with sodium pentobarbital (20 mg/kg IV).10,11 The animals were then intubated and ventilated with room air, and oxygen was supplemented via a positive-pressure respirator (Shinano Inc). Arterial pH, Po2, and Pco2 were kept within normal ranges. Under aseptic conditions, a left thoracotomy was performed and the proximal segments of the left anterior and circumflex coronary arteries were carefully dissected. The following three protocols were performed.
The dissected segment of the coronary artery was gently wrapped with cotton mesh absorbing 0.05 mL of IL-1β (2.5 μg) suspension alone for immunostaining. The time course of the expression of eNOS and iNOS was examined before (n=2) and 1 (n=4), 3 (n=4), and 14 (n=2) days after the surgery.
The three dissected coronary segments were gently wrapped with cotton mesh absorbing one of the following suspensions in 9 pigs: (1) 0.05 mL of sepharose beads bound to recombinant human IL-1β (2.5 μg) alone, (2) IL-1β (2.5 μg)+aminoguanidine (a selective inhibitor of iNOS, 0.25 mg),22,23 and (3) IL-1β (2.5 μg)+L-NAME (a nonspecific inhibitor of both eNOS and iNOS, 250 mg). Angiographic study was performed 1 day and 2 weeks after the operation.
The three dissected coronary segments were gently wrapped with cotton mesh absorbing one of the following suspensions in 8 pigs: (1) 0.05 mL of sepharose beads bound to recombinant human IL-1β (2.5 μg) alone, (2) L-NAME (250 mg) alone, and (3) IL-1β (2.5 μg)+L-NAME (250 mg). In another 3 pigs, the coronary segment was similarly treated with aminoguanidine (0.25 mg) alone. Angiographic study was performed 2 weeks after the operation.
These different treatments were performed at different sites in the coronary arteries. The treated sites of the coronary arteries were randomized. In a previous study, we confirmed that the applied cytokine did not affect either the vasomotor responses or the histology of the adjacent coronary segment, which ruled out the possibility of cytokine leakage from the cotton mesh.10,11
We tested different doses of aminoguanidine, 10 and 100 times less (2.5 and 0.25 mg) than the dose of L-NAME (250 mg), and found that 0.25 mg of aminoguanidine selectively inhibited iNOS alone and that 2.5 mg of aminoguanidine inhibited both iNOS and eNOS in our model. Thus, the cotreatment with L-NAME (250 mg) inhibited both eNOS and iNOS, whereas the cotreatment with aminoguanidine (0.25 mg) inhibited iNOS alone in our in vivo preparation.
This experiment was reviewed by the Ethics Committee on Animal Experiment at the Kyushu University School of Medicine and was carried out in accordance with the Guidelines for Animal Experiment at the Kyushu University School of Medicine and The Law (No. 105) and Notification (No. 6) of the Japanese Government.
Preparation of Cytokine Beads
Sepharose microbeads (1 g; CNBr-activated sepharose 4B, 45 to 165 μm in diameter, Pharmacia), which bind to the amino residues of proteins, including cytokines, were added to 50 mL of 1 mmol HCl solution and centrifuged four times at 1200 rpm for 5 minutes each time.10,11 The beads were then resuspended in 20 mL of NaHCO3/NaCl solution with 1 mg of cytokines (IL-1β). The beads were allowed to bind with the cytokines at room temperature for 1 hour and then at 4°C overnight. After centrifugation at 1200 rpm for 5 minutes, the supernatant was separated, and the concentration of the remaining cytokine in the supernatant was measured by an ELISA.10,11 The cytokine-bound beads in the pellet were resuspended with Tris/HCl buffer solution for 1 hour to block any remaining active sites. The cytokine-bound beads were finally washed and resuspended so that the final concentration of cytokine was 50 μg/mL. The number of cytokines or control beads in the suspension was ≈70/μL. All of the above procedures were performed under sterile conditions.10,11
Because in our bead preparation most of the IL-1β molecules were bound inside the beads by a covalent bond at the amino residues of the protein, ≤1.2% of the IL-1β molecules were actually bound to the surface of the beads and biologically active. Thus, when 2.5 μg of IL-1β bound to the beads was applied to the coronary artery, ≤30 ng of IL-1β was biologically active.10,11 In addition, we previously confirmed that the treatment with control beads alone causes minimal intimal thickening and no hyperconstrictive responses.10,11
Immunohistochemical Staining of the Coronary Artery
In protocol 1, the hearts were excised without angiography to avoid any possible influences of the procedure on the expression of eNOS and iNOS. The left coronary artery was then perfused with physiological saline (1000 mL) at a pressure of 120 mm Hg, and the tissue samples (cytokine-treated and untreated portions) were immediately embedded in OCT compound, sectioned at a thickness of 4 μm, and mounted on glass slides. After rehydration, immunoenzymatic staining was performed.24 The sections were preincubated with 0.1% Triton-X in PBS and 0.1% skim milk to reduce the occurrence of nonspecific reactions. Antibodies to eNOS, iNOS, and nonimmune IgG (negative control) were applied and incubated for 60 minutes at room temperature.24 The sections were incubated for biotinylated anti-mouse rabbit immunoglobulin for 10 minutes and then incubated with 1% hydrogen peroxide in methanol to reduce the occurrence of nonspecific reactions to peroxidase. Then the sections were incubated with peroxidase-labeled streptavidin solution for 10 minutes. The slides were rinsed in PBS with 0.1% Triton-X after each incubation step. The peroxidase activity was determined with DAB buffer tablets (Merck 64271) with hydrogen peroxide (0.013%). The slides were counterstained with hematoxylin solution, dehydrated, and mounted.24 The extent of the expression of eNOS and iNOS in the coronary artery was evaluated by two observers in a blinded manner.
In protocol 2, coronary arteriography was performed 1 day after the surgery to examine the nonspecific inhibitory effect of L-NAME for both eNOS and iNOS and the selective inhibitory effect of aminoguanidine for iNOS alone. The animals were sedated with ketamine hydrochloride (12.5 mg/kg IM) and then were anesthetized with sodium pentobarbital (20 mg/kg IV). The animals were then intubated and ventilated with room air while oxygen was supplemented via a positive-pressure respirator. Heparin (3000-U bolus IV) was administered every 60 minutes. First, control coronary arteriography was performed. Then, the coronary vascular responses were examined in response to the intracoronary administration of bradykinin (10 and 100 ng/kg), UK14304, a selective α2-adrenergic agonist25 (1, 3, and 10 μg/kg), d-arginine (0.1 and 1 μg/kg), and l-arginine (0.01, 0.1, and 1 μg/kg). Coronary arteriography was performed 2 minutes after intracoronary administration of bradykinin and UK14304 and 3 minutes after that of d-arginine and l-arginine, when the vasodilator effect of each agent peaked.
In both protocols 2 and 3, coronary arteriography was performed 2 weeks after the surgery to examine the coronary vasospastic responses. Coronary arteriography was first performed before and 2 minutes after the intracoronary administration of nitroglycerin (10 μg/kg). Then, the coronary vascular responses were examined in response to the intracoronary administration of serotonin (10 μg/kg). Coronary arteriography was performed 2 minutes after intracoronary administration of serotonin.10,11
Each dose of drugs was diluted with 1 mL of physiological saline and was injected into the left coronary artery. The same amount of saline was used to flush the catheter. Throughout the study, the catheter position remained fixed.10,11
Coronary Arteriography and Hemodynamic Measurements
The animals were anesthetized and ventilated as described above, and selective coronary arteriography was performed.10,11 A preshaped Judkins catheter was inserted into the right or left femoral artery, and then coronary arteriography in a left anterior oblique view was performed. ECGs in leads I, II, III, V1, and V6 were recorded. The arterial pressure was measured with a pressure transducer (Gould Inc) connected to the Kifa catheter. The arterial pressure, heart rate, and ECGs were continuously monitored and recorded on a pen recorder (NEC San-Ei Polygraph System).10,11
Selective coronary arteriography was performed in a left anterior oblique projection that provided a clear visualization of the cytokine-treated sites with the Toshiba cineangiography system (KXO-1250/CAS-CA, Toshiba Medical Inc). The angiograms were recorded on 35-mm cinefilm (Varicath I, VARI-X) at 48 frames per second. The angle of the projection, the position of the animal, and the distance from the x-ray focus to the animal and that from the animal to the image intensifier were all carefully kept constant during each experiment.10,11
The cineangiograms were projected on a screen with a cineprojector (ELK-35CB, Nishimoto Sangyo Inc), and an end-diastolic frame was selected. The coronary luminal diameters were measured with a caliper.10,11 With this technique, excellent correlations between repeated measurements (r=.99) and between different observers (r=.98) were confirmed in the range of the coronary diameter from 0.98 to 5.58 mm.10,11 The degree of the constrictive response was expressed as the percent decrease in the luminal diameter from the control level. The luminal diameters were measured at the sites treated with IL-1β, L-NAME, IL-1β+L-NAME, aminoguanidine, or IL-1β+aminoguanidine. The diameters of the treated coronary segments were all comparable (Table⇓).
The animals were then euthanized with a lethal dose of sodium pentobarbital and exsanguinated, and then the heart was excised. The left coronary artery was perfused with 6% formalin at a pressure of 120 mm Hg and fixed with formalin for 1 week. For the light microscopic examination, tissue samples were embedded in paraffin, sectioned into slices 5 μm thick, mounted on glass slides, and stained with hematoxylin-eosin and van Gieson’s method.10,11
With a photomicroscopic photograph system (Microphot-FXA, Nikon Co), pictures of the coronary arteries were taken at magnifications of ×40 and ×100. Each specimen was then evaluated for the presence of intimal proliferation, luminal encroachment, medial dissection, and any alteration of the internal or external elastic lamina.10,11
The degree of the intimal thickening was analyzed quantitatively with a computer-assisted picture analysis system (Genlocker System, Sony).10,11 This system consists of a high-resolution television monitor, an image processing and calculation unit with a microprocessor, a light pen controller with a microprocessor, and a printer. The inner border of the intimal layer and the internal elastic lamina were traced by a light pen, and the areas encircled by the tracing were calculated automatically. The intimal area (Ai) was calculated by the formula Ai=Ae−Al, where Ae and Al are the areas within the internal elastic lamina and the internal border of the vessel (luminal area) at ×40 magnification.10,11 The degree of the intimal thickening was expressed by the percent intima area (Ai/Ae×100%).10,11
The following drugs were used: antibodies to eNOS (H32, a generous gift from Dr Pollock, Abbott Laboratory), iNOS (PA3–030, Affinit and Bioreagents), UK14304 (a selective α2-agonist, Pfizer Central Research), and bradykinin, l-arginine, d-arginine, 5-hydroxytryptamine (serotonin), and nitroglycerin (Sigma Chemical Co).
All results are expressed as the mean±SEM. The differences in organic stenosis and intimal area were evaluated by one-way ANOVA followed by Fisher’s test for multiple comparisons. When serial changes in the coronary artery diameter in response to drugs were compared, two-way ANOVA followed by Fisher’s test was used for multiple comparisons. A value of P<.05 was considered to be statistically significant.
Immunohistochemistry for eNOS and iNOS
Immunohistochemical staining showed that at the IL-1β–treated site, eNOS in the endothelium was maintained throughout the experimental period, whereas iNOS was markedly induced 1 day after the operation, mainly in the coronary smooth muscle, and thereafter downregulated until 14 days (Fig 1⇓).
Confirmation of the Inhibitory Effects of L-NAME and Aminoguanidine In Vivo
Hemodynamic variables and coronary artery diameters 1 day and 2 weeks after the operation are shown in the Table⇑.
One day after the operation, intracoronary bradykinin caused mild coronary vasodilation at the untreated site, whereas UK 14304 caused little coronary vasodilation at the same site. However, both endothelium-dependent vasodilators caused significant coronary vasodilation at the site treated with either IL-1β alone or IL-1β+aminoguanidine (Fig 2⇓). In contrast, the vasodilation was abolished at the site treated with IL-1β+L-NAME (Fig 2⇓). Intracoronary l-arginine caused coronary vasodilatation at the IL-1β–treated site, which was abolished at the site treated with IL-1β+aminoguanidine or with IL-1β+L-NAME (Fig 2⇓). Conversely, intracoronary d-arginine caused no relaxation at any site (Fig 2⇓).
Two weeks after the application of IL-1β, the augmented coronary dilation to either bradykinin or UK 14304 was no longer noted at the site treated with IL-1β alone or with IL-1β+aminoguanidine (Fig 3⇓). Similarly, the coronary vasodilation to intracoronary l-arginine was no longer noted at the IL-1β–treated site, and d-arginine again caused no relaxation at any site (Fig 3⇓).
The extent of the coronary dilating response to intracoronary nitroglycerin (20 μg/kg) did not differ significantly among the treated sites either 1 day or 2 weeks after the application of IL-1β (data not shown).
Chronic Effects of L-NAME and Aminoguanidine on the IL-1β–Induced Coronary Hyperconstrictive Responses
Two weeks after the operation, intracoronary serotonin caused coronary vasoconstriction at the IL-1β–treated site, which tended to be greater at the sites treated with IL-1β+aminoguanidine and with IL-1β+L-NAME (Fig 4⇓). The summarized data are shown in Fig 5⇓. The cotreatment with aminoguanidine tended to augment and that with L-NAME significantly augmented the IL-1β–induced hyperconstrictive responses; however, no difference was noted between the effects of the two inhibitors (Fig 5A⇓). Compared with the untreated segment, the coronary vasoconstriction to serotonin was greater at the site treated with L-NAME alone but not at the site treated with aminoguanidine alone (Fig 5B⇓).
Chronic Effects of L-NAME and Aminoguanidine on the IL-1β–Induced Neointimal Formation
A histological examination revealed the development of neointimal formation at the IL-1β–treated site. The extent of the neointimal formation was significantly greater at the site treated with IL-1β+aminoguanidine and with IL-1β+L-NAME than at the site treated with IL-1β alone (Fig 6⇓). The cotreatment with aminoguanidine significantly aggravated the IL-1β–induced neointimal formation, whereas no further aggravation was noted with the cotreatment with L-NAME (Fig 6A⇓). Neointimal formation was also noted at the site treated with IL-1β alone but not at the site treated with aminoguanidine alone (Fig 6B⇓).
The novel findings of the present study were that (1) treatment with IL-1β induced a transient expression of iNOS in the coronary smooth muscle in vivo, (2) eNOS in endothelial cells was functionally maintained at the IL-1β–treated site in vivo, and (3) NO produced by iNOS appears to exert an inhibitory effect on the IL-1β–induced proliferative/vasospastic changes of the coronary artery in vivo. To the best of our knowledge, this is the first demonstration that the transient production of NO by iNOS plays an important vasculoprotective role against the inflammatory cytokine-induced proliferative/vasospastic changes of the coronary artery in vivo.
Inflammatory Cytokines and the NOS System of the Coronary Artery
Although inflammatory cytokines are not expressed in the normal artery, they are induced in atherosclerotic lesions and cause the release of a number of growth factors/cytokines from vascular wall cells in the cytokine network, resulting in the proliferation of vascular smooth muscle cells.6 Indeed, we recently demonstrated that chronic treatment with IL-1β induces arteriosclerotic and vasospastic changes of the coronary artery in vivo.10,11
Studies in vitro have demonstrated that the exposure of vascular smooth muscle cells to such inflammatory cytokines as IL-1β is associated with the release of a large amount of NO due to the induction of iNOS in vascular smooth muscle cells.12-14,16 Indeed, in the present study we confirmed histologically that iNOS was transiently but intensely expressed in response to the IL-1β stimulation. We also confirmed that this iNOS expression was functionally associated with increased coronary vasodilating response to l-arginine. We thus examined whether or not the transient production of NO by iNOS has a deleterious (as in the case in cardiac myocytes) or protective effect on the coronary artery in vivo.
Inhibition of eNOS and iNOS in the Coronary Artery In Vivo
In the present animal study, we inhibited both eNOS and iNOS in the coronary artery using a relatively high dose of L-NAME and inhibited iNOS alone with a relatively low dose of aminoguanidine in vivo. Aminoguanidine has been shown to selectively inhibit the cytokine-induced iNOS in vitro.22,23 Its hydrazine moiety may be important for its selectivity for iNOS because replacement with methyl groups (methylguanidine and 1,1-dimethylguanidine) results in a loss of selectivity.26 Aminoguanidine is equipotent to L-NMMA as an inhibitor of the cytokine-induced isoforms of NOS but is 10- to 100-fold less potent as an inhibitor of eNOS.22,23,26 The dose of aminoguanidine required to produce a comparable degree of increase in arterial blood pressure is 16 to 40 times higher than that of L-NMMA.22,23,26 The arteriography study 1 day after the operation confirmed these findings in our model in vivo. However, it should be noted that the low dose of aminoguanidine (0.25 mg) may not completely inhibit iNOS activity. More importantly, however, the present dose of aminoguanidine apparently did not affect the eNOS activity in vivo. We thus used the present dose of aminoguanidine to dissect the actions of eNOS and iNOS in vivo, whereas we used a relatively high dose of L-NAME to completely inhibit the entire vascular NOS system.
IL-1β–Induced Expression of iNOS in the Coronary Artery
The present animal study demonstrated that the treatment with IL-1β indeed induces the expression of iNOS in vascular smooth muscle cells of the coronary artery in vivo. The expression of iNOS was transient, however; the expression peaked 1 day after the operation and thereafter decreased until 14 days. This time course may correspond to that of IL-1β activity, which disappeared 14 days after the operation in our model.10,11 The induction of iNOS in the vascular smooth muscle is also known to be inhibited in vitro by glucocorticoids,13 transforming growth factor-β,27 and angiotensin II.28 Thus, the iNOS induction may also be reduced in vivo under the conditions in which the effects of those endogenous vasoactive substances are augmented.
In contrast to the previous in vitro finding with TNF-α,21 in the present study the expression of eNOS was well maintained throughout the study period, exerting its inhibitory effect on the IL-1β–induced changes of the coronary artery. The reason for the difference between our in vivo findings and the previous in vitro findings remains to be clarified.
IL-1β may also induce other enzyme systems that can have important vascular effects, such as COX 2.29,30 However, we have recently found in a preliminary study that NS 398, a selective and potent inhibitor of COX 2,31 does not affect the IL-1β–induced vasospastic and arteriosclerotic changes of the coronary artery, suggesting that the contribution of COX 2 in our model with IL-1β may be minimal (unpublished observations). Possible alterations in other vascular enzyme systems induced by IL-1β remain to be examined in a future study.
Inhibitory Effects of iNOS on the IL-1β–Induced Neointimal Formation
Studies in vitro have shown that NO-generating vasodilators inhibit the proliferation of vascular smooth muscle cells.32 Recent studies in vivo also demonstrated that the chronic administration of l-arginine33-35 reduces and the chronic inhibition of NO synthesis aggravates36,37 the neointimal formation after arterial balloon injury. However, these studies failed to dissect the relative roles of eNOS and iNOS. iNOS is known to be transiently induced in response to arterial injury.38 The present study demonstrated for the first time that iNOS, which is transiently induced and produces a large amount of NO in response to arterial injury, plays a more important role than eNOS in suppressing the neointimal formation. The relative importance of iNOS compared with eNOS may be due to the difference in the amount of NO that the two NOS isoforms can produce in the acute phase after arterial injury.16 However, the present study also suggests the important role of eNOS because the treatment with L-NAME alone but not with aminoguanidine alone caused the development of mild hyperconstriction to serotonin and neointimal formation. Endothelial covering and the continuous release of NO from endothelial cells both appear to inhibit the platelet/leukocyte adhesion and aggregation and hence the subsequent initiation of the proliferative processes.19,20
Inhibitory Effects of iNOS on the IL-1β–Induced Coronary Hyperconstrictive Responses
The present study demonstrated that the IL-1β–induced hyperconstrictive responses to serotonin were aggravated at the site cotreated with aminoguanidine or L-NAME 2 weeks after the IL-1β application. At this time, iNOS activity was absent and eNOS activity was fairly maintained. Thus, the transient production of a large amount of NO by iNOS shortly after the cytokine application appeared to inhibit the development of the vasospastic responses by inhibiting the coronary arteriosclerotic changes.
We previously demonstrated that the pathway mediated by protein kinase C in medial smooth muscle cells plays an important role in the pathogenesis of coronary spasm in our original swine model with coronary spasm39 as well as in our present model with IL-1β.40 We also have recently demonstrated that phenotypes of medial smooth muscle cells are altered toward dedifferentiation41 and that myosin light chain phosphorylations in medial smooth muscle cells are markedly enhanced on stimulation by serotonin.42 We consider that NO produced by iNOS inhibited the arteriosclerotic changes that are associated with an upregulation of the protein kinase C–mediated pathway in medial smooth muscle cells, which results in the inhibition of the coronary hyperconstrictive responses in vivo.
We have recently demonstrated in vivo that different types of arterial injury induced by IL-1β and balloon angioplasty similarly cause the proliferative/vasospastic responses via the cytokine/growth factor network.10,11,43 These two types of vascular injury are known to induce iNOS in vascular smooth muscle.12-14,38 In addition, iNOS is also induced in atherosclerotic lesions locally and repetitively by inflammatory cytokines.6 The findings of the present study thus suggest the potential importance of the therapeutic strategy to locally increase the iNOS expression while at the same time indicating the potentially harmful role of a suppression of iNOS expression13,27,28 for the prevention of coronary vascular events and restenosis after vascular injury.
Selected Abbreviations and Acronyms
|COX 2||=||cyclooxygenase 2|
|eNOS||=||endothelial nitric oxide synthase|
|iNOS||=||inducible nitric oxide synthase|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|NOS||=||nitric oxide synthase|
|TNF-α||=||tumor necrosis factor-α|
This work was supported in part by grants from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare, Tokyo, Japan, and grants-in-aid from the Sandoz Foundation for Gerontological Research, Basel, Switzerland, the Japan Research Foundation for Clinical Pharmacology, Tokyo, Japan, and the Japanese Medical Association, Tokyo, Japan. The authors wish to thank Drs T. Yamawaki and K. Miyata for cooperation in this study and T. Takebe, M. Mizokami, and K. Fujii for their excellent technical assistance.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.
- Received June 3, 1997.
- Revision received August 4, 1997.
- Accepted August 13, 1997.
- Copyright © 1997 by American Heart Association
Maseri A, Severi S, DeNes DM, L’Abbate A, Chierchia S, Marzilli M, Ballestra AM, Parodi O, Biagini A, Distante A. ‘Variant’ angina: one aspect of a continuous spectrum of vasospastic myocardial ischemia: pathogenetic mechanisms, estimated incidence and clinical and coronary angiographic findings in 138 patients. Am J Cardiol. 1978;42:1019-1035.
Shimokawa H, Tomoike H, Nabeyama S, Yamamoto H, Araki H, Nakamura M, Ishii Y, Tanaka K. Coronary artery spasm induced in atherosclerotic miniature swine. Science. 1983;221:560-562.
Egashira K, Tomoike H, Yamamoto Y, Yamada A, Hayashi Y, Nakamura M. Histamine-induced coronary spasm in regions of intimal thickening in miniature pigs: roles of serum cholesterol and spontaneous or induced intimal thickening. Circulation. 1986;74:826-837.
Hannson GK, Jonasson L, Seifert PS, Stemme S. Immune mechanisms in atherosclerosis. Arteriosclerosis. 1989;9:567-578.
Libby P, Warner SJC, Friedman G. Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest. 1988;81:487-498.
Shimokawa H, Ito A, Fukumoto Y, Kadokami T, Nakaike R, Sakata M, Takayanagi T, Egashira K, Takeshita A. Chronic treatment with interleukin-1β induces coronary intimal lesions and vasospastic responses in pigs in vivo: the role of platelet-derived growth factor. J Clin Invest. 1996;97:769-776.
Ito A, Shimokawa H, Kadokami T, Fukumoto Y, Owada MK, Shiraishi T, Nakaike R, Takayanagi T, Egashira K, Takeshita A. Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1β in pigs in vivo. J Clin Invest. 1995;96:1288-1294.
Shibano T, Vanhoutte PM. Induction of NO production by TNF-α and lipopolysaccharide in porcine coronary arteries without endothelium. Am J Physiol. 1993;264:H403-H407.
Kanno K, Hirata Y, Imai T, Marumo F. Induction of nitric oxide synthase gene by interleukin in vascular smooth muscle cells. Hypertension. 1993;22:34-39.
Tsujino M, Hirata Y, Imai T, Kanno K, Eguchi S, Ito H, Marumo F. Induction of nitric oxide synthase gene by interleukin-1β in cultured rat cardiocytes. Circulation. 1994;90:375-383.
Moncada S, Higgs EA. Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB J. 1995;9:1319-1330.
Yoshizumi M, Perrella MA, Burnett JC Jr, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. J Biol Chem. 1993;73:205-209.
Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem. 1981;29:577-580.
Shimokawa H, Flavahan NA, Vanhoutte PM. Loss of endothelial pertussis toxin—sensitive G-protein function in atherosclerotic porcine coronary arteries. Circulation. 1991;83:652-660.
Corbett JA, Tilton RG, Chang K, Hasan KS, Ido Y, Wang JI, Sweetland MA, Lancaster JR, Williamson JR, McDaniel MI. Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes. 1992;41:552-556.
Kanno K, Hirata Y, Imai T, Iwashina M, Marumo F. Regulation of inducible nitric oxide synthase gene by interleukin-1β in rat vascular endothelial cells. Am J Physiol. 1994;267:H2318-H2324.
Nakayama I, Lawahara Y, Tsuda T. Angiotensin II inhibits cytokine-stimulated inducible nitric oxide synthase expression in vascular smooth muscle cells. J Biol Chem. 1994;269:11628-11633.
Coyne DW, Nickols M, Bertland W, Morrison AR. Regulation of mesangial cell cyclooxygenase synthesis by cytokines and glucocorticoids. Am J Physiol. 1992;263:F97-F102.
Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1777.
Hamon M, Vallet B, Bauters C, Wernert N, McFadden EP, Lablance JM, Dupuis B, Bertrand ME. Long-term oral administration of l-arginine reduces intimal thickening and enhances neoendothelium-dependent acetylcholine-induced relaxation after arterial injury. Circulation. 1994;90:1357-1362.
Tarry WC, Makhoul RG. l-Arginine improves endothelium-dependent vasorelaxation and reduces intimal hyperplasia after balloon angioplasty. Arterioscler Thromb. 1994;14:938-943.
Naruse K, Shimizu K, Muramatsu M, Toki M, Miyazaki Y, Okumura K, Hashimoto H, Ito T. Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta: PGH2 does not contribute to impaired endothelium-dependent relaxation. Arterioscler Thromb. 1994;14:746-752.
Cayatte AJ, Palacino JJ, Hotton K, Cohen RA. Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits. Arterioscler Thromb. 1994;14:753-759.
Joly GA, Schini VB, Vanhoutte PM. Balloon injury induces nitric oxide synthase activity in rat carotid arteries. J Cardiovasc Pharmacol. 1992;20:S151-S154.
Ito A, Shimokawa H, Nakaike R, Fukai T, Sakata M, Takayanagi T, Egashira K, Takeshita A. Role of protein kinase C–mediated pathway in the pathogenesis of coronary artery spasm in a swine model. Circulation. 1994;90:2425-2431.
Kadokami T, Shimokawa H, Fukumoto Y, Ito A, Takayanagi T, Egashira K, Takeshita A. Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C–mediated pathway in a swine model with interleukin-1β in vivo. Circulation. 1996;94:190-196.
Fukumoto Y, Shimokawa H, Ito A, Kadokami T, Yonemitsu Y, Aikawa M, Owada MK, Egashira K, Sueishi K, Nagai R, Yazaki Y, Takeshita A. Inflammatory cytokines cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs. J Cardiovasc Pharmacol. 1997;29:222-231.
Katsumata N, Shimokawa H, Seto M, Kozai T, Yamawaki T, Kuwata K, Egashira K, Ikegaki I, Asano T, Sasaki Y, Takeshita A. Enhanced myosin light chain phosphorylations as a central mechanism for coronary artery spasm with interleukin-1β. Circulation. In press.
Fukumoto Y, Shimokawa H, Kozai T, Kadokami T, Kuwata K, Owada MK, Shiraishi T, Kuga T, Egashira K, Takeshita A. Tyrosine kinase inhibitor suppresses the (re)stenotic changes of the coronary artery after balloon injury in pigs. Cardiovasc Res. 1996;32:1131-1140.