© 1996 American Heart Association, Inc.
Articles |
Expression of Inducible Nitric Oxide Synthase Inhibits Platelet Adhesion and Restores Blood Flow in the Injured Artery
From the Department of Medicine and King Gustaf V Research Institute (Z.Y., T.Y., G.K.H.) and the Department of Physiology and Pharmacology (W.Z.), Karolinska Institute, Stockholm, Sweden.
Correspondence to Prof Goran K. Hansson, King Gustaf V Research Institute, Karolinska Hospital, S-17176 Stockholm, Sweden.
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Abstract |
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NO generated by endothelial cells is vasoprotective by antagonizing platelet adhesion and smooth muscle contraction. Since vascular smooth muscle cells (VSMCs) produce NO in response to cytokine stimulation and after arterial injury, we speculated that NO produced by VSMCs could compensate for the loss of endothelium. Using balloon catheter denudation of the rat carotid artery as a model for arterial injury and restenosis, we have evaluated the time course of expression of inducible NO synthase (iNOS) by in situ hybridization and immunohistochemistry and studied the effect of iNOS on platelet adhesion and blood flow of the injured vessel. iNOS mRNA and protein were expressed in the innermost layer of the media from day 1 and were subsequently detected in the neointima, whereas no expression was detectable in the uninjured artery. Systemic administration of N
-nitro-L-arginine methyl ester (L-NAME) resulted in a twofold to threefold increase in the adhesion of 111In-labeled platelets to the injured vessel wall. Platelet adhesion was also enhanced threefold by local delivery of L-NAME from a gel surrounding the injured vessel, whereas the stereoisomer, D-NAME, had no effect. Finally, inhibition of NO synthase led to a 24% reduction of the blood flow in the injured carotid artery. These results demonstrate that arterial injury triggers the expression of iNOS in the lesion and that NO produced by iNOS inhibits platelet adhesion and restores blood flow. This could explain the disappearance of platelet thrombi from deendothelialized arterial surfaces within a few days after injury and indicates the importance of NO generated by iNOS for the maintenance of vascular tone. Thus, expression of iNOS in lesions may represent a protective mechanism that compensates for the loss of endothelium.
Key Words: angioplasty • nitric oxide • platelet • blood flow • vascular smooth muscle
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nitric oxide is an important intercellular and intracellular signal molecule that regulates the cardiovascular, nervous, and immune systems (see References 1 and 2 for review). It is generated from the terminal guanidino nitrogen group of L-arginine by the enzyme NOS. Two isoforms of NOS have been identified in the cardiovascular system.3 4 5 6 Under physiological conditions, a constitutive Ca2+-dependent NOS in endothelial cells (eNOS or NOS3) is responsible for the continuous release of NO.7 In the presence of proinflammatory cytokines, such as interleukin-1, IFN-
, and tumor necrosis factor-
, or bacterial endotoxin, an inducible Ca2+-independent NOS (iNOS) is expressed in VSMCs, leading to persistent generation of large quantities of NO.8 9 10 11 NO produced by endothelial cells plays a key role in the maintenance of vascular tone, the regulation of VSMC proliferation, and the control of platelet and leukocyte adhesion to the endothelium.12 However, the pathophysiological consequences of iNOS expression in injured arteries are less clear. Injury of a vascular segment elicits a complex sequence of events characterized by platelet adhesion and aggregation, leukocyte infiltration, and smooth muscle cell migration and proliferation (see References 13 and 14 for review). In experimental models, platelet aggregation and adhesion to the wall of injured vessels has been demonstrated in the early phase after balloon injury.15 However, it is noteworthy that platelets disappear from the arterial surface within a few days after injury, although reendothelialization does not occur for several weeks or even months.16 17 This implies the activation of a mechanism that inhibits platelet adhesion and thrombus formation on the injured vascular surface.
It has recently been demonstrated that iNOS is rapidly induced in neointimal VSMCs after arterial injury and that the induction of iNOS at the sites of injury is accompanied by significant NO production.18 Because of its important biological properties, we speculated that NO derived from iNOS in the injured vessel wall may function as a modulator of restenosis and atherosclerosis. Therefore, the present study was designed (1) to characterize the expression and distribution of iNOS in the rat carotid artery in response to balloon injury and (2) to determine whether NO derived from VSMC iNOS modulates platelet adhesion and local hemodynamics of the injured vessel. Our results indicate that iNOS is induced in the arterial media within 1 day after injury and that inhibition of NOS activity enhances platelet adhesion to the injured arterial surface and reduces local blood flow.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat Injury Model
Male Sprague-Dawley rats (average weight, 400 g; 3 to 4 months old) were used in the present study, which was approved by the regional ethical committee for animal research. All surgical procedures were performed under general anesthesia by an intraperitoneal injection of flunisonium/fentanyl. The left carotid artery was denuded as described previously.19 Briefly, a Fogarty F2 balloon catheter (Baxter Healthcare Corp) was introduced through the left external carotid artery and advanced into the aorta; the balloon was inflated with 0.2 mL of air and then withdrawn to the entry point. The entire procedure was repeated three times, and the external carotid artery was ligated after removal of the catheter. For studying expression and distribution of iNOS, animals were killed under flunisonium/fentanyl anesthesia at the indicated times after injury (from 1 day to 1 week) and perfused with 100 mL PBS via the left ventricle. Tissues were snap-frozen in liquid nitrogen and kept at -80°C for immunohistochemistry and in situ hybridization.
Pharmacological Treatment
Immediately after the balloon procedure, animals (five to seven rats per group) were injected subcutaneously with L-NAME (Sigma Chemical Co) at a dose of 50 mg/kg body wt daily from the day of surgery and onwards. Control rats were injected with an identical volume of physiological saline solution. For local delivery of drugs, a 25% (wt/vol) solution of Pluronic F-127 (BASF-Wyandotte Corp) with or without L-NAME or its inactive enantiomer, D-NAME, were prepared and maintained at 4°C. After ballooning, 100 µL of Pluronic F-127 solution containing 0.1 or 1.0 mg L-NAME or 1.0 mg D-NAME was immediately applied and allowed to form a gel surrounding the exposed arterial segments. In control animals, denudation was performed as described above, but either an "empty" Pluronic F-127 gel or no gel at all was applied to the perivascular tissue.
Analysis of Platelet Adhesion
Platelets were labeled with 111In-oxine using a modified method.20 Briefly, 18 mL of blood from two littermate donor rats was collected in tubes containing 2 mL of ACD. The blood was centrifuged for 15 minutes at 200g to prepare platelet-rich plasma, from which platelets were recovered by centrifugation at 640g for 10 minutes. The platelet-poor plasma supernatant was collected, and the platelet pellet was washed and suspended in 1 mL of diluted ACD. Platelets were incubated with 1 mCi 111In-oxine (Amersham) for 20 minutes at room temperature, and an 111In-labeled platelet pellet was obtained by centrifugation at 640g for 10 minutes, washed with diluted ACD, and resuspended in platelet-poor plasma for injection. The labeling efficiency was 
70% according to the following equation:
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111In-labeled (0.4 mL) platelet suspension was administered to each animal via the tail vein 1 hour before death. The rats were killed by exsanguination under flunisonium/fentanyl anesthesia and perfused via the left ventricle with 100 mL of 1% paraformaldehyde in 15 mmol/L phosphate buffer (pH 7.2). A defined 15-mm segment of the denuded distal left artery and the corresponding segment of the right carotid artery were excised and analyzed for 111In radioactivity with a gamma counter.
Assessment of Blood Flow in Carotid Artery
Rats were anesthetized with flunisonium/fentanyl. Body temperature was continuously monitored with a rectal thermometer and kept at 37°C. Blood flow of the left carotid artery was measured with a Transonic T206 Flowmeter (Transonic System Inc), and the mean blood pressure was measured with a Micro Switch 156 PC pressure transducer (Micro Switch) connected with a model 7D Grass polygraph (Grass Instrument Co).
Determination of Platelet cGMP
Arterial blood samples were collected from the animals 3 days after balloon injury and anticoagulated with 5 mmol/L EDTA. Platelet-rich plasma was assayed for cGMP using a commercially available radioimmunoassay kit (Amersham). Platelets in platelet-rich plasma were counted with a Coulter counter.
In Situ Hybridization Analysis
Antisense and sense oligonucleotide probes corresponding to nucleotides 561 to 600 of the rat VSMC iNOS cDNA5 were synthesized on an Applied Biosystems PCR-Mate oligonucleotide synthesizer. The probes were 3'-end labeled with digoxigenin by using an oligonucleotide tailing kit (Boehringer Mannheim). Labeling was performed according to the manufacturer's instruction, except that the labeling reaction time was reduced to 15 minutes to minimize probe digestion, and the final ethanol wash was omitted. The hybridization buffer contained 5x SSC, 50% formamide, 0.15 mg/mL yeast tRNA, and 2x Denhardt's solution. Prehybridization for 30 minutes at room temperature was followed by overnight hybridization at 42°C with 200 ng of either the antisense or the sense probe. Slides were rinsed briefly in 1x SSC, then washed in 1x SSC/0.1% SDS three times for 20 minutes each at 52°C, and washed once with 1x SSC/0.1% SDS for 20 minutes at a temperature that dropped from 52°C to room temperature. For detection of hybridization, sections were then incubated with a monoclonal antibody to digoxigenin (Boehringer Mannheim), followed by rabbit anti-mouse immunoglobulin, an alkaline phosphatase-antialkaline phosphatase complex (Dakopatts), and nitro blue tetrazolium/5-bromo-4-chloro-3-indoxyl phosphate substrate solution.
Immunohistochemistry
Serial cryostat sections (7-µm) were air-dried, fixed for 10 minutes in cold acetone at -20°C, preincubated with 5% fat-free dry milk to block nonspecific antibody binding, and then incubated overnight at 4°C with polyclonal rabbit antibodies against murine macrophage iNOS (Affinity BioReagents) diluted 1:500 with PBS. Subsequently, sections were incubated with a biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 minutes at room temperature, followed by incubation with avidin-biotin-alkaline phosphatase complexes (Vector Laboratories) for 30 minutes at room temperature and the chromogenic alkaline phosphatase substrate for another 20 minutes. Control staining was performed by replacing the primary antibody with nonimmune rabbit serum.
Statistical Analysis
Results are reported as mean±SEM. Student's t test was used to evaluate differences between means, and values of P<.05 were considered statistically significant.
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Results |
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Expression of VSMC iNOS in the Injured Carotid Artery
Expression of VSMC iNOS mRNA after balloon injury to the rat carotid artery was characterized by in situ hybridization and immunohistochemistry. Fig 1A
shows that hybridization of the antisense probe to VSMC iNOS mRNA was observed in the sections obtained 1 day after balloon injury. Since iNOS mRNA is not expressed in uninjured arteries,18 this indicates a rapid induction of iNOS mRNA in response to arterial injury. The hybridization signal was also present in sections obtained 3 days after balloon injury (Fig 1B). iNOS mRNA was predominantly localized to VSMCs in the innermost layer of the media at these early stages (Fig 1A and 1B). Control hybridization with the sense probe was negative (Fig 1C).
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To characterize iNOS expression on the protein level, specimens of injured arteries were immunostained with polyclonal antibodies against iNOS. This showed that iNOS protein was present in a pattern essentially identical to that observed by in situ hybridization. Thus, iNOS could be observed in the innermost layer of the media as early as 1 day after balloon injury (Fig 1D and 1E
). Control immunostaining, in which the primary antibody was replaced with normal rabbit serum, produced no positive signal (Fig 1F).
In situ hybridization analysis of lesions at 5 to 7 days after injury revealed iNOS mRNA in the forming neointima as well as in the innermost layer of the media (Fig 2A and 2B). Uninjured carotid arteries from normal rats showed no detectable iNOS mRNA signal (not shown). Immunostaining indicated that VSMCs of the neointima and inner media expressed iNOS protein and that the expression was particularly abundant among VSMCs at the vascular surface (Fig 2C and 2D).
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Effect of NOS Inhibition on Platelet Adhesion
An in vivo adhesion assay was used to characterize platelet deposition on the forming lesion. 111In-labeled platelets were injected intravenously 1 hour before the rats were killed, and radioactivity was measured in the injured segment. Fig 3, top, shows that platelet deposition on the injured arterial segment was time dependent, with maximal 111In activity during the first day after injury, followed by decreasing 111In activity during the subsequent 5 days.
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To determine the role of endogenous NO in platelet deposition, L-NAME, a specific inhibitor of NOS, was administered systemically at a daily dose of 50 mg/kg. This resulted in increased platelet adhesion to the injured arterial segment (Fig 3, top), whereas it did not increase adhesion to the corresponding intact segment of the contralateral carotid artery (Fig 3, bottom).
Since NOS is found in platelets,21 22 systemic administration of L-NAME might exert generalized effects on platelet function, in addition to any effects on vascular NOS. To specifically inhibit NOS activity in the arterial wall, we used a local delivery system for the administration of L-NAME. The inhibitor was suspended in a Pluronic F-127 solution that was subsequently polymerized perivascularly in the living animal. This resulted in a slow release of L-NAME from the gel to the arterial tissue, until the Pluronic F-127 gel was dissolved after 2 days (data not shown). Platelet adhesion assays were performed 3 days after balloon injury. As shown in Fig 4, the Pluronic F-127 gel alone did not have any effect on platelet adhesion to the injured arterial wall. In contrast, L-NAME delivered locally via the gel drastically increased platelet adhesion. Compared with the empty gel control, locally administered L-NAME at 0.1 and 1.0 mg augmented platelet-derived 111In activity in the injured arterial segment 2.3- and 4.5-fold, respectively (Fig 4). Unlike L-NAME, the inactive enantiomer, D-NAME, had no significant effect (Fig 4).
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Effect of NOS on Blood Flow of the Injured Carotid Artery
To evaluate the hemodynamic effect of iNOS, the blood flow of the carotid artery was measured before, 10 minutes after, and 3 days after vascular injury. As shown in the Table, vascular injury resulted in a 9% to 16% reduction of blood flow. The reduction of blood flow in the injured artery was transient, since it was restored to the preinjury level after 3 days in the two control groups. However, animals treated systemically or locally with L-NAME still showed a 25% and 32% decrease in blood flow, respectively, compared with their preinjury levels. Since L-NAME could totally prevent the restoration of blood flow in the injured vessels, this indicates that iNOS activity in the lesion plays an important role in modulating the local hemodynamics.
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Platelet cGMP Level
To assess whether NO produced by iNOS in the lesion could affect the reactivity of circulating platelets, platelet cGMP levels in arterial blood were determined 3 days after denudation of the carotid artery. In comparison with normal animals, platelet cGMP was not significantly affected in the injured rats (Fig 5). Moreover, local delivery of L-NAME had no significant effect on platelet cGMP, whereas systemic administration of L-NAME caused an 18% reduction of the cGMP level. These results indicate that iNOS expressed in the injured carotid artery has no significant effect on cGMP levels in circulating platelets.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO is an important regulator of vascular tone and nonthrombogenity.1 23 It is normally produced by NOS expressed in the endothelium, and inhibition of the enzyme results in vasoconstriction and platelet adhesion.24 25 26 Deendothelialization results in the immediate adhesion of platelets to the injured vessel wall,15 16 but larger thrombi do not form, and the platelets disappear from the injured surface within a few days.16 17 However, the mechanisms that counteract thrombus formation in injured arteries have been unclear.
We now demonstrate that deendothelializing injury is followed by expression of iNOS in VSMCs and that the ensuing NO production inhibits platelet adhesion and restores blood flow in the injured vessel. Our results suggest that the expression of iNOS in response to injury provides a homeostatic mechanism to control thrombus formation and to regulate local hemodynamics in the injured artery.
Our conclusions are based on the detection of iNOS expression in injured arteries and on an analysis of platelet adhesion and local blood flow under conditions of NO production and its inhibition. Combined in situ hybridization and immunohistochemical studies with oligonucleotides and antibodies specific for iNOS showed that this gene product is rapidly induced in medial VSMCs after balloon catheter injury. iNOS expression persisted during the subsequent week, when VSMCs migrate from the media into the intima, where they proliferate to form an elevated lesion.13 14
Platelet adhesion to the injured artery was highest on the first day after injury and then gradually declined during the subsequent week. Administration of an NOS inhibitor, L-NAME, increased platelet adhesion severalfold, indicating that enzymatic production of NO modulates platelet adhesion to the injured arterial surface. This is further supported by the striking effect of L-NAME and complete lack of effect of the inactive enantiomer, D-NAME, on the platelet deposition.
The data from the local delivery experiment demonstrate that local NO production in the injured artery is important for the nonthrombogenic effect. The pronounced effect of L-NAME on platelet deposition and the concomitant expression of iNOS and reestablishment of nonthrombogenity strongly argue for an important role of iNOS in the protection against thrombosis after vascular injury. This view is also supported by the recent observation that platelet deposition on injured vessels is enhanced by removal of NO with hemoglobin or by inhibition of NOS and prevented by L-arginine treatment.27
In addition to its antiplatelet action, NO has profound effects on vascular contractility. In the present study, NO production in the injured artery was important for the restoration of blood flow in the injured carotid artery. This, in turn, should reduce the risk for development of clinically significant restenosis.
The present morphological analysis of iNOS expression confirms and extends our previous report on iNOS expression in injured arteries.18 We now show that iNOS is expressed by medial VSMCs as early as 24 hours after balloon injury and persists during medial proliferation, migration into the neointima, and neointimal proliferation. In addition, we demonstrate that transcription of the iNOS gene is accompanied by accumulation of iNOS protein in VSMCs in the injured artery.
Although the present studies provide evidence that iNOS-dependent NO production correlates with the appearance of nonthrombogenity, inhibition of enzymatic activity could not totally prevent the nonthrombogenic process at later stages. This indicates that NO exerts a dominant antiplatelet role in the early phase and that other mechanisms are involved at later stages. It is known that the antiplatelet activity of NO is due largely to its interaction with soluble guanylate cyclase, which results in an increased cGMP concentration in the platelet.28 29 30 The subsequent cGMP-mediated reactions are less clear, but they inhibit the expression of platelet glycoprotein IIb/IIIa and P-selectin.31 32 However, the results indicating that expression of iNOS restores the local blood flow and that NO generated from iNOS in the injured carotid artery has no significant effect on cGMP in circulating platelets suggest that platelet deposition is regulated by a confined interaction between deposited platelets and subendothelium. NO synergizes with other platelet inhibitory mediators, such as prostacyclin and carbon monoxide,33 34 35 36 37 and fibrinolytic mechanisms may also be important.38 Obviously, the nonthrombogeneity of the vascular surface depends on the combined effects of NO, prostacyclin, and other mechanisms.33 34 35 36 37 38
The early induction of iNOS expression is surprising, and its triggering mechanisms remain unclear. In cell culture, the proinflammatory cytokines, interleukin-1, IFN-, and tumor necrosis factor-, synergistically induce iNOS in cultured rat VSMCs.7 8 9 39 However, in rat arteries, leukocyte adhesion and infiltration are very limited during the first days after injury. Therefore, it is unlikely that induction of iNOS during this early stage is triggered by cytokines released from activated inflammatory cells. Instead, recent reports that shear stress response elements are present in the promoter region of the iNOS gene40 led us to speculate that cytokine-independent mechanisms activated during tissue injury might induce iNOS expression on the first day after injury. Furthermore, it is noteworthy that the expression of iNOS in VSMCs coincides with the phenotypic modulation, migration, and proliferative response of these cells after injury.41 42 43 One might therefore surmise that all these phenomena could be controlled by the same factors. Further studies will be necessary to determine whether cytokines and/or other mechanisms are responsible for iNOS induction following arterial injury.
In conclusion, the results of the present study demonstrate that (1) iNOS is rapidly induced in the deendothelialized artery in response to balloon injury, (2) that iNOS-expressing VSMCs are initially localized in the inner media and then migrate to the neointima, (3) that NOS-dependent NO production inhibits platelet adhesion to the denuded artery, and (4) that it restores blood flow in the denuded arterial segment. Together, these results unveil a homeostatic mechanism that compensates for the loss of endothelium by inducing expression of iNOS in the VSMC population of the artery during the response to injury.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Swedish Medical Research Council (Projects 6816 and 4764), the Swedish Heart-Lung Foundation, the Nanna Svartz Fund, and the King Gustaf V 80th Anniversary Fund. Dr Yokota was the recipient of a postdoctoral stipend from the Swedish Institute and a Medical Research Council fellowship.
Received October 23, 1995; accepted April 5, 1996.
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References |
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 C.-Y. Wei, K.-C. Huang, Y.-H. Chou, P.-F. Hsieh, K.-H. Lin, and W.-W. Lin The Role of Rho-Associated Kinase in Differential Regulation by Statins of Interleukin-1beta- and Lipopolysaccharide-Mediated Nuclear Factor {kappa}B Activation and Inducible Nitric-Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells Mol. Pharmacol., March 1, 2006; 69(3): 960 - 967. [Abstract] [Full Text] [PDF]
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 Z. W. Q. Moore and D. Y. Hui Apolipoprotein E inhibition of vascular hyperplasia and neointima formation requires inducible nitric oxide synthase J. Lipid Res., October 1, 2005; 46(10): 2083 - 2090. [Abstract] [Full Text] [PDF]
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A. Sato, K. Terata, H. Miura, K. Toyama, F. R. Loberiza Jr., O. A. Hatoum, T. Saito, I. Sakuma, and D. D. Gutterman Mechanism of vasodilation to adenosine in coronary arterioles from patients with heart disease Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1633 - H1640. [Abstract] [Full Text] [PDF]
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 M. Zanetti, L. V. d'Uscio, I. Kovesdi, Z. S. Katusic, and T. O'Brien In Vivo Gene Transfer of Inducible Nitric Oxide Synthase to Carotid Arteries From Hypercholesterolemic Rabbits Stroke, May 1, 2003; 34(5): 1293 - 1298. [Abstract] [Full Text] [PDF]
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P. Vermeersch, Z. Nong, E. Stabile, O. Varenne, H. Gillijns, M. Pellens, N. Van Pelt, M. Hoylaerts, I. De Scheerder, D. Collen, et al. L-Arginine Administration Reduces Neointima Formation After Stent Injury in Rats by a Nitric Oxide-Mediated Mechanism Arterioscler Thromb Vasc Biol, October 1, 2001; 21(10): 1604 - 1609. [Abstract] [Full Text] [PDF]
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 W. Gosgnach, D. Messika-Zeitoun, W. Gonzalez, M. Philipe, and J.-B. Michel Shear stress induces iNOS expression in cultured smooth muscle cells: role of oxidative stress Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1880 - C1888. [Abstract] [Full Text] [PDF]
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 D. Behr-Roussel, A. Rupin, S. Simonet, E. Bonhomme, S. Coumailleau, A. Cordi, B. Serkiz, J.-N. Fabiani, and T. J. Verbeuren Effect of Chronic Treatment With the Inducible Nitric Oxide Synthase Inhibitor N-Iminoethyl-L-Lysine or With L-Arginine on Progression of Coronary and Aortic Atherosclerosis in Hypercholesterolemic Rabbits Circulation, August 29, 2000; 102(9): 1033 - 1038. [Abstract] [Full Text] [PDF]
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 G. Valen, G. K Hansson, A. Dumitrescu, and J. Vaage Unstable angina activates myocardial heat shock protein 72, endothelial nitric oxide synthase, and transcription factors NF{kappa}B and AP-1 Cardiovasc Res, July 1, 2000; 47(1): 49 - 56. [Abstract] [Full Text] [PDF]
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R. Muniyappa, R. Xu, J. L. Ram, and J. R. Sowers Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1762 - H1768. [Abstract] [Full Text] [PDF]
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 M. Shimpo, U. Ikeda, Y. Maeda, K.-i. Ohya, Y. Murakami, and K. Shimada Effects of Aspirin-Like Drugs on Nitric Oxide Synthesis in Rat Vascular Smooth Muscle Cells Hypertension, May 1, 2000; 35(5): 1085 - 1091. [Abstract] [Full Text] [PDF]
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B. Jiang and P. Brecher N-Acetyl-L-Cysteine Potentiates Interleukin-1{beta} Induction of Nitric Oxide Synthase : Role of p44/42 Mitogen-Activated Protein Kinases Hypertension, April 1, 2000; 35(4): 914 - 918. [Abstract] [Full Text] [PDF]
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Z.-q. Yan, A. Sirsjo, M.-L. Bochaton-Piallat, G. Gabbiani, and G. K. Hansson Augmented Expression of Inducible NO Synthase in Vascular Smooth Muscle Cells During Aging Is Associated With Enhanced NF-{kappa}B Activation Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2854 - 2862. [Abstract] [Full Text] [PDF]
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B. Jiang, M. Haverty, and P. Brecher N-Acetyl-L-Cysteine Enhances Interleukin-1{beta}-Induced Nitric Oxide Synthase Expression Hypertension, October 1, 1999; 34(4): 574 - 579. [Abstract] [Full Text] [PDF]
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M. Kibbe, T. Billiar, and E. Tzeng Inducible nitric oxide synthase and vascular injury Cardiovasc Res, August 15, 1999; 43(3): 650 - 657. [Abstract] [Full Text] [PDF]
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U. Ikeda, M. Ikeda, S. Minota, and K. Shimada Homocysteine Increases Nitric Oxide Synthesis in Cytokine-Stimulated Vascular Smooth Muscle Cells Circulation, March 9, 1999; 99(9): 1230 - 1235. [Abstract] [Full Text] [PDF]
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T. Le Tourneau, E. Van Belle, D. Corseaux, B. Vallet, G. Lebuffe, B. Dupuis, J.-M. Lablanche, E. McFadden, C. Bauters, and M. E. Bertrand Role of nitric oxide in restenosis after experimental balloon angioplasty in the hypercholesterolemic rabbit: effects on neointimal hyperplasia and vascular remodeling J. Am. Coll. Cardiol., March 1, 1999; 33(3): 876 - 882. [Abstract] [Full Text] [PDF]
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 J. Binko, S. Meachem, and H. Majewski Endothelium removal induces iNOS in rat aorta in organ culture, leading to tissue damage Am J Physiol Endocrinol Metab, January 1, 1999; 276(1): E125 - E134. [Abstract] [Full Text] [PDF]
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M. Iwashina, M. Shichiri, F. Marumo, and Y. Hirata Transfection of Inducible Nitric Oxide Synthase Gene Causes Apoptosis in Vascular Smooth Muscle Cells Circulation, September 22, 1998; 98(12): 1212 - 1218. [Abstract] [Full Text] [PDF]
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S. Chandra, L. V. Clark, R. W. Coatney, L. Phan, S. K. Sarkar, and E. H. Ohlstein Application of Serial In Vivo Magnetic Resonance Imaging to Evaluate the Efficacy of Endothelin Receptor Antagonist SB 217242 in the Rat Carotid Artery Model of Neointima Formation Circulation, June 9, 1998; 97(22): 2252 - 2258. [Abstract] [Full Text] [PDF]
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S. Janssens, D. Flaherty, Z. Nong, O. Varenne, N. van Pelt, C. Haustermans, P. Zoldhelyi, R. Gerard, and D. Collen Human Endothelial Nitric Oxide Synthase Gene Transfer Inhibits Vascular Smooth Muscle Cell Proliferation and Neointima Formation After Balloon Injury in Rats Circulation, April 7, 1998; 97(13): 1274 - 1281. [Abstract] [Full Text] [PDF]
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U. Ikeda, Y. Maeda, H. Funayama, Y. Hojo, M. Ikeda, S. Minota, S. Kano, and K. Shimada Monocyte-vascular smooth muscle cell interaction enhances nitric oxide production Cardiovasc Res, March 1, 1998; 37(3): 820 - 825. [Abstract] [Full Text] [PDF]
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 Z.-q. Yan and G. K. Hansson Overexpression of Inducible Nitric Oxide Synthase by Neointimal Smooth Muscle Cells Circ. Res., January 23, 1998; 82(1): 21 - 29. [Abstract] [Full Text] [PDF]
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G. Vassalli and D. A Dichek Gene therapy for arterial thrombosis Cardiovasc Res, September 1, 1997; 35(3): 459 - 469. [Abstract] [Full Text] [PDF]
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C. Greenlees, R. M Wadsworth, P. A Martorana, and C. L Wainwright The effects of L-arginine on neointimal formation and vascular function following balloon injury in heritable hyperlipidaemic rabbits Cardiovasc Res, August 1, 1997; 35(2): 351 - 359. [Abstract] [Full Text] [PDF]
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U. Ikeda, K. Kurosaki, K.-i. Ohya, and K. Shimada Adenosine stimulates nitric oxide synthesis in vascular smooth muscle cells Cardiovasc Res, July 1, 1997; 35(1): 168 - 174. [Abstract] [Full Text] [PDF]
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C. A. E. O'Blenes, C. Kinnear, and M. Rabinovitch Tumor Necrosis Factor-{alpha} Induces Fibronectin Synthesis in Coronary Artery Smooth Muscle Cells by a Nitric Oxide-Dependent Posttranscriptional Mechanism Circ. Res., July 6, 2001; 89(1): 26 - 32. [Abstract] [Full Text] [PDF]
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