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Vasculoprotective roles of neuronal nitric oxide synthase ¹

TSUYOSHI MORISHITA^*, MASATO TSUTSUI^*,2, HIROAKI SHIMOKAWA, MASATAKA HORIUCHI^*, AKIHIDE TANIMOTO, OSAMU SUDA^*, HIROMI TASAKI^*, PAUL L. HUANG, YASUYUKI SASAGURI, NOBUYUKI YANAGIHARA and YASUHIDE NAKASHIMA^*

^* Second Department of Internal Medicine, Departments of
Pharmacology and
Pathology and Cell Biology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan;
Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; and
Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

²Correspondence: Second Department of Internal Medicine, University of Occupational and Environmental, Health, School of Medicine, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: mt2498{at}med.uoeh-u.ac.jp

SPECIFIC AIM

Neuronal nitric oxide synthase (nNOS) exists in the neointima, endothelial cells, and macrophages in both early and advanced atherosclerotic lesions in humans. However, little is known about the role of nNOS expressed in those vascular lesions. Thus, we addressed this point in two different animal models of vascular injury.

PRINCIPAL FINDINGS

1. nNOS gene-deficient (nNOS-KO) mice exhibit accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption in vivo
We first examined the effect of a targeted deletion of nNOS gene on vascular lesion formation in a model of unilateral carotid artery ligation in mice. In the control carotid artery, no pathological finding was noted in either the wild-type or nNOS-KO mice (Fig. 1 A). By contrast, in the ligated carotid artery, neointimal formation and constrictive vascular remodeling (defined as reduction in vascular cross-sectional area) were noted 4 wk after the procedure in both strains (Fig. 1A ). The extent of neointimal formation (as evaluated by the intima-to-media ratio and wall thickness in the ligated carotid artery), of constrictive vascular remodeling (as evaluated by reduction in external elastic lamina length in the ligated carotid to the control carotid artery), and of luminal narrowing (as evaluated by the lumen diameter in the ligated carotid artery) were all significantly accelerated in the nNOS-KO mice compared with wild-type mice (Fig. 1A, B ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Histology of mouse carotid artery. A) Hematoxylin-eosin staining. EEL, external elastic lamina; IEL, internal elastic lamina. B) Quantitative analysis of vascular lesions (n=9, *P<0.05). WT, wild-type mice; nNOS-KO, nNOS-KO mice.

2. nNOS is up-regulated in vascular lesions caused by blood flow disruption in mice
Immunostaining revealed that nNOS protein was absent in the control and the ligated carotid artery of the nNOS-KO mice and in the control carotid artery of wild-type mice. By contrast, on days 3 and 7 after carotid artery ligation, nNOS immunoreactivity was noted in infiltrating inflammatory cells at the adventitia, most of which were neutrophils. On days 14 and 28, nNOS immunoreactivity was localized predominantly in the neointima and medial smooth muscle cells and, to a lesser extent, in endothelial cells.

3. Selective inhibition of nNOS activity enhances vasoconstrictor responses to a variety of calcium-mobilizing stimuli and suppresses tissue cGMP concentrations in balloon-injured arteries in rats
We next examined whether functional up-regulation of nNOS is involved in a rat balloon injury model. In the control carotid arteries with endothelium, selective nNOS inhibitors SMTC and 7-NI did not significantly affect angiotensin II-induced contractions (Fig. 2 A). By contrast, in the balloon-injured carotid arteries at 4 wk after the procedure, SMTC and 7-NI significantly augmented the angiotensin II-induced contractions (Fig. 2B ). A nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) and a selective iNOS inhibitor aminoguanidine (AG) also enhanced angiotensin II-induced contractions in the balloon-injured arteries (Fig. 2C ). The angiotensin II-induced contractions of the balloon-injured arteries were inhibited by AT₁ receptor antagonist losartan but not by AT₂ receptor antagonist PD123319 (Fig. 2D ).

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of NOS inhibitors or angiotensin II receptor antagonists on angiotensin II-induced contractions in rat carotid arteries. A) SMTC (10 µM, n=6), 7-NI (10 µM, n=5). B) SMTC (10 µM, n=6), 7-NI (10 µM, n=8). C) L-NAME (300 µM, n=4), AG (3 mM, n=6). D) Losartan (100 nM, n=3), PD123319 (1 µM, n=3). *P < 0.05.

Stimulation with angiotensin II (30 nM) did not change tissue cGMP concentrations (a marker of vascular NO production) in the control arteries with endothelium, but significantly increased them in the balloon-injured arteries. SMTC and 7-NI as well as L-NAME significantly reduced the basal and the angiotensin II-induced increase in tissue cGMP concentrations in the balloon-injured arteries.

SMTC and 7-NI did not significantly affect the contractions to KCl and endothelin-1 in the control arteries. By contrast, both significantly enhanced contractions to those vasoconstrictors in the balloon-injured arteries.

4. Selective inhibition of nNOS activity exacerbates neointimal formation in balloon-injured arteries in rats
Long-term oral treatment with 7-NI for 14 days significantly exacerbated neointimal formation and reduced lumen diameter in balloon-injured arteries.

5. nNOS is induced in vascular wall cells after balloon injury in rats
The immunoreactivity for nNOS was absent in the control artery but was observed faintly in the neointima on day 7 after balloon injury and became evident on day 28 not only in the neointima, but also in medial smooth muscle cells. Western blot analysis showed nNOS protein expression only in the balloon-injured arteries.

CONCLUSIONS

The novel findings of the present study were as follows. 1) nNOS-KO mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption, 2) selective inhibition of nNOS activity significantly enhanced various vasoconstrictor responses, suppressed vascular cGMP production, and exacerbated neointimal formation in rat balloon-injured arteries, and 3) in both models, nNOS was up-regulated in vascular lesions after injury. To the best of our knowledge, this is the first report that demonstrates the important vasculoprotective role of nNOS in the vascular lesion formation in vivo.

We have previously demonstrated that NO derived from eNOS inhibits neointimal formation whereas NO derived from iNOS suppresses the development of constrictive vascular remodeling in the same carotid ligation model, using eNOS-KO and iNOS-KO mice. The present study revealed that NO derived from nNOS also suppresses neointimal formation and constrictive vascular remodeling. These results indicate that all three NOS isoforms have different vasculoprotective actions against vascular lesion formation in vivo.

In the early stage of vascular lesion formation (days 3 and 7 after the carotid artery ligation), nNOS immunoreactivity was first noted in neutrophils, especially at the adventitia, suggesting that nNOS inhibits the development of constrictive vascular remodeling (Fig. 3 ). In the advanced stage of vascular lesion formation (days 14 and 28 after the procedure), nNOS was expressed in the neointima, medial smooth muscle cells, and endothelial cells, suggesting that nNOS in those cells suppresses neointimal formation (Fig. 3) .

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the functional significance of nNOS induced in two animal models of vascular injury. In the early stage of vascular lesion formation in the mouse ligation model, expression of nNOS was noted at the adventitial inflammatory cells (mostly neutrophils), suggesting that nNOS inhibits the development of constrictive vascular remodeling. In the advanced stage of vascular lesion formation, nNOS was expressed in the neointima, medial smooth muscle cells, and endothelial cells, suggesting that such nNOS suppresses neointimal formation. In rat balloon injury model, increased concentrations of intracellular calcium in vascular smooth muscle cells stimulated by angiotensin, II, KCl, and endothelin-1 may cause not only vasocontractions, but also activation of nNOS, which results in inhibition of contractions via NO and subsequent cGMP generation. Moreover, up-regulation of nNOS appears to inhibit neointimal formation in the rat model. Thus, nNOS may play important vasculoprotective roles in vascular lesion formation in vivo.

We next examined whether the functional up-regulation of nNOS is involved in a rat balloon injury model. Selective nNOS inhibitors, SMTC and 7-NI, as well as a nonselective NOS inhibitor, L-NAME, significantly enhanced angiotensin II-induced contractions in the balloon-injured arteries but not in control arteries. Angiotensin II-induced contractions of the balloon-injured arteries were inhibited by AT₁ receptor antagonist losartan but not by AT₂ receptor antagonist PD123319. Stimulation with angiotensin II did not change tissue cGMP concentrations, a marker of vascular NO production, in the control arteries with endothelium, but significantly increased them in the balloon-injured arteries. SMTC and 7-NI, as well as L-NAME, significantly reduced the basal and the angiotensin II-induced increase in tissue cGMP concentrations in the balloon-injured arteries. nNOS was absent in the control artery but noted in neointimal and medial smooth muscle cells in the balloon-injured arteries. These results indicate that nNOS is functionally up-regulated in the neointima and the media after balloon injury in vivo, exerting an important inhibitory effect on the AT₁ receptor-mediated vasoconstrictor responses by enhancing the NO/cGMP-mediated pathway (Fig. 3) .

Selective inhibition of nNOS activity with SMTC and 7-NI enhanced the contractions to KCl and endothelin-1 as well as those to angiotensin II in balloon-injured arteries. It is well established that angiotensin II, KCl, and endothelin-1 cause a sustained and large increase in intracellular calcium concentrations in vascular smooth muscle cells (VSMC). Therefore, the increased concentrations of intracellular calcium in VSMC stimulated by those agonists may result in vasoconstriction and NO generation through calcium-dependent activation of nNOS, with a subsequent increase in tissue cGMP concentrations (Fig. 3) .

Long-term oral treatment with 7-NI significantly exacerbated neointimal formation and luminal narrowing in balloon-injured arteries in rats. This provides additional evidence that nNOS suppresses neointimal formation in the rat model of balloon injury (Fig. 3) , which partially mimics the nNOS-KO mice phenotype. Under pathological conditions associated with elevated intracellular calcium concentrations in vascular smooth muscle, such as arteriosclerosis and vasospasm, nNOS may serve as an alternative source of NO, inhibiting vascular lesion formation and spastic responses in an autocrine/paracrine manner.

In conclusion, our findings provide the first direct evidence that nNOS is expressed after vascular injury in vivo and plays an important vasculoprotective role. Up-regulation of nNOS may thus play an important compensatory role in the presence of reduced eNOS activity (e.g., inflammation and arteriosclerosis) to maintain vascular homeostasis in vivo.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0155fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0155fje

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