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(Circulation Research. 2004;95:822.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Structure of Cerebral Arterioles in Mice Deficient in Expression of the Gene for Endothelial Nitric Oxide Synthase

Gary L. Baumbach, Curt D. Sigmund, Frank M. Faraci

From the Departments of Pathology (G.L.B.) and Internal Medicine (C.D.S., F.M.F.), University of Iowa College of Medicine & Cardiovascular Center, Iowa City, Iowa.

Correspondence to Gary L. Baumbach, MD, Department of Pathology, 5231D RCP, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail g-baumbach{at}uiowa.edu

	Abstract

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We examined effects of pharmacological inhibition of nitric oxide synthase (NOS) and genetic deficiency of the endothelial isoform of NOS (eNOS) on structure and mechanics of cerebral arterioles. We measured pressure, diameter, and cross-sectional area (CSA) of the vessel wall (histologically) in maximally dilated cerebral arterioles in mice that were untreated or treated for 3 months with the NOS inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg per day in drinking water). Treatment with L-NAME increased systemic arterial mean pressure (SAP; 143±4 versus 121±4 mm Hg, P<0.05) and CSA (437±27 versus 310±34 µm², P<0.05). These findings suggest that hypertension induced in mice by NOS inhibition is accompanied by hypertrophy of cerebral arterioles. To determine the role of the eNOS isoform in regulation of cerebral vascular growth, we examined mice with targeted disruption of one (heterozygous) or both (homozygous) genes encoding eNOS. Wild-type littermates served as controls. SAP and CSA were significantly increased in homozygous (SAP, 141±5 versus 122±3 mm Hg in wild-type mice, P<0.05; CSA, 410±18 versus 306±15 µm² in wild-type mice, P<0.05), but not in heterozygous (SAP, 135±4 mm Hg; CSA, 316±32 µm²) eNOS-deficient mice. Carotid ligation normalized cerebral arteriolar pulse pressure did not prevent increases in CSA in homozygous eNOS-deficient mice. Thus, cerebral arterioles undergo hypertrophy in homozygous eNOS-deficient mice, even in the absence of increases in arteriolar pulse pressure. These findings suggest that eNOS plays a major role in regulation of cerebral vascular growth.

Key Words: hypertension • L-NAME • endothelial nitric oxide synthase deficiency • vascular hypertrophy

	Introduction

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Small resistance arteries and arterioles undergo hypertrophy in several models of chronic hypertension.^1–3 Several determinants apparently contribute to vascular hypertrophy, including increases in arterial pressure,⁴ particularly pulse pressure,^5–7 sympathetic nerves,^8,9 genetic factors,³ the renin-angiotensin system,^10,11 and the endothelium-derived contracting factor, endothelin.¹² Another determinant that may contribute to vascular hypertrophy during chronic hypertension is nitric oxide (NO). This suggestion is based on the following observations. First, it has been observed that vasodilator drugs that generate NO inhibit mitogenesis and proliferation of vascular muscle in culture.¹³ Second, we have found that hypertension induced in rats with the nitric oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), produced hypertrophy of cerebral arterioles, even when increases in cerebral arteriolar pulse pressure were attenuated with unilateral carotid ligation.¹⁴ These observations suggest that in addition to regulation of resting vascular resistance, NO may play an important role in regulation of vascular structure.

A major limitation of using NOS inhibitors to study the role of NO in cerebral vascular hypertrophy is that most of these agents nonselectively inhibit all isoforms of NOS. Multiple isoforms of NOS may be expressed within the vessel wall, and there are no known selective inhibitors of NOS. For example, in addition to the endothelial isoform of NOS (eNOS), neuronal NOS (nNOS) is expressed in vascular muscle.¹⁵ Furthermore, both perivascular nerves and brain tissue contain large amounts of nNOS,^16,17 and nNOS has been shown to influence cerebral vascular tone^18,19 and may therefore also influence cerebral vascular growth. Thus, regardless of the outcome of studies using a NOS inhibitor, no definitive conclusions regarding its effects on cerebral vascular structure and mechanics can be drawn in relation to the specific isoforms of NOS involved. Mice with targeted disruption of the gene encoding for eNOS^20,21 provide an opportunity to examine the role of eNOS in complex physiological systems. Through studies performed in eNOS-deficient mice, the role of eNOS can be defined without relying on pharmacological inhibitors that lack specificity for NOS isoforms.

The first goal of this study was to examine effects of chronic treatment with L-NAME on structure and mechanics of cerebral arterioles in mice. Based on our previous findings in rats treated with L-NAME,¹⁴ we anticipated that cerebral arterioles in L-NAME–treated mice would undergo hypertrophy. The second goal was to test the hypothesis that cerebral arterioles undergo hypertrophy in mice lacking the gene for eNOS. In addition to providing an approach for examining the role of the eNOS isoform in cerebral vascular structure and mechanics, using eNOS-deficient mice in this study also addresses other potential limitations of L-NAME treatment. For example, NOS inhibitors may stimulate vascular hypertrophy through an interaction with angiotensin II AT₁ receptors.^22–24

	Materials and Methods

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Animals
Experiments were conducted on male C57BL/6J mice and male and female eNOS-deficient mice. Animals were allowed free access to food and tap water, housed at 25°C, and exposed to 12 hours of light each day. Procedures followed in this study were in accordance with institutional guidelines for care and use of experimental animals at the University of Iowa.

L-NAME–Treated Mice
Male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Me). To examine effects of inhibition of NO synthase on cerebral arterioles, mice were treated with L-NAME (1 g/L) in the drinking water beginning at 4 weeks of age. Mice that drank tap water served as controls. Water intake in the L-NAME–treated group was 200 mL/kg per day. Thus, intake of L-NAME during the treatment period was 200 mg/kg per day. Cerebral arterioles were examined after 3 months of treatment.

eNOS-Deficient Mice
eNOS-deficient mice were produced as described previously.²¹ We generated eNOS^+/+, eNOS^+/–, and eNOS^–/– mice by interbreeding eNOS^+/– mice that had been derived from 7 to 9 generations of backcross breeding to C57BL/6J mice. This approach allowed us to use eNOS^+/+ littermates as controls. With littermate controls, any heterogeneity in genetic background, on average, will be similar in both control and genetically altered mice. We included the study of eNOS^+/– mice, because it has been observed previously that vascular responses to some stimuli are altered in eNOS^+/– mice.^25,26 In addition, experts in the field of genetics encourage the study of heterozygous deficient mice because, depending on the results, they may encourage the study of genetic polymorphisms in humans.²⁷ Genotyping of the animals was performed by Southern blotting or polymerase chain reaction from tail biopsy specimens as previously described.^25,26,28,29

To determine whether effects of eNOS deficiency on cerebral arterioles resulted from increases in arterial pressure, separate groups of eNOS^+/+ and eNOS^–/– mice (obtained from Jackson Laboratories) underwent unilateral carotid ligation at 4 weeks of age. Mice were anesthetized with Avertin (0.2 to 0.3 mL, intraperitoneally), shaved, and prepared with a 70% alcohol solution, and a ligature was tied tightly around the left common carotid artery. To control for possible damage to sympathetic nerves by the carotid ligature, a second ligature was tied loosely around the right carotid artery.

In Vivo Preparation
We examined structure of cerebral arterioles in 6-month-old L-NAME–treated mice and 6-month-old eNOS-deficient mice. Because anesthesia can lower arterial pressure in mice, particularly in mice with chronic hypertension, we measured systemic arterial pressure in conscious mice using a method we have described previously.^30,31 After measurement of conscious arterial pressure, animals were weighed and anesthetized with sodium pentobarbital (5 mg 100 g^–1 body weight intraperitoneally), intubated, and mechanically ventilated with room air and supplemental O₂. Additional anesthesia (1.7 mg 100 g^–1 body weight intravenous) was administered when pressure to a paw evoked a change in blood pressure or heart rate.

A catheter was inserted into a femoral vein for injection of drugs and fluids. A catheter was inserted into a femoral artery to record systemic arterial pressure and obtain blood samples for measurement of arterial blood gases, and a catheter was inserted into the other femoral artery to withdraw blood to produce hypotension (needed for studies of vascular mechanics).

Measurement of Cerebral Arteriolar Pressure and Diameter
We measured pressure and diameter in first-order arterioles on the cerebrum³² through an open skull preparation that we have described in detail previously.^30,31 In mice with unilateral carotid ligation, a dam of acrylic was constructed along the exposed portion of the superior sagittal suture, and craniotomies were made over parietal cortex of the left and right cerebral hemispheres. Cerebral arteriolar systolic, diastolic, mean, and pulse pressures were measured continuously with a micropipette connected to a servo-null pressure-measuring device (model 5; Instrumentation for Physiology and Medicine, Inc).

Arterioles were monitored through a microscope connected to a closed-circuit video system with a final magnification of x356. Images of arterioles were digitized using a video frame grabber installed in a Macintosh computer (Quadra 900; Apple Computer). Arteriolar diameter was measured from the digitized images by the use of image analysis software (NIH Image; National Institutes of Health, Research Services Branch, National Institute of Mental Health).

Experimental Protocol
Approximately 20 to 30 minutes after completion of surgery, measurements of cerebral arterioles were obtained under baseline conditions. Vascular smooth muscle was then deactivated by suffusion of cerebral vessels with artificial cerebral spinal fluid containing EDTA (67 mmol/L), which produces complete deactivation of smooth muscle in cerebral arterioles.³³ Pressure–diameter relationships were obtained in deactivated cerebral arterioles between cerebral arteriolar pressures of 40 and 10 mm Hg. Maximally dilated arterioles were fixed at physiological pressure in vivo by suffusion of vessels with glutaraldehyde fixative (2.25% glutaraldehyde in 0.10 mol/L cacodylate buffer) while maintaining cerebral arteriolar pressure at baseline levels. Arterioles were considered to be adequately fixed when blood flow through the arteriole had ceased. After the anesthetized animal was euthanized by an injection of sodium pentobarbital, the arteriolar segment used for pressure–diameter measurements was removed, processed for electron microscopy, and embedded in Spurr low-viscosity resin while maintaining cross-sectional orientation. Cross-sectional area of the arteriolar wall was determined histologically using a method we have described in detail previously.^6,33

Statistical Analysis
Analysis of variance was used to compare systemic mean pressure, arteriolar pressures, diameters, cross-sectional area of the vessel wall, and slope of tangential elastic modulus versus stress. Probability values were calculated using a Student t test. Statistics were determined using JMP statistics software (SAS Institute Inc) on a Macintosh computer.

	Results

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L-NAME–Treated Mice
Systemic mean arterial pressure was significantly greater in L-NAME–treated mice than in untreated mice in both the unanesthetized and anesthetized states, even though anesthesia significantly reduced systemic mean arterial pressure in both groups (Table 1). Cerebral arteriolar systolic, diastolic, mean, and pulse pressures were significantly greater in L-NAME–treated mice than in untreated mice (Table 1). Diameter before deactivation with EDTA was not significantly different in cerebral arterioles of mice treated with L-NAME than in untreated mice (Table 1). During deactivation with EDTA, internal and external diameters and cross-sectional area of the vessel wall were greater in cerebral arterioles in mice treated with L-NAME than in cerebral arterioles in untreated mice (Table 1). Thus, during L-NAME–induced hypertension in mice, cerebral arterioles underwent hypertrophy of the vessel wall.

View this table: [in this window] [in a new window]   Table 1. Baseline Values in L-NAME–Treated Mice

Vascular Mechanics
Internal diameter in cerebral arterioles during maximal dilatation was larger in L-NAME–treated mice than in untreated mice at all levels of arteriolar pressure between 10 and 40 mm Hg (Figure 1, left panel). The stress–strain curve in cerebral arterioles in L-NAME–treated mice was similar to the curve in cerebral arterioles in control mice (Figure 1, right panel). In addition, the slope of tangential elastic modulus versus stress was not significantly different in L-NAME–treated mice and untreated mice (Table 1). These findings suggest that L-NAME–induced hypertension was not accompanied by alterations in passive distensibility of cerebral arterioles, despite hypertrophy of the vessel wall.

View larger version (13K): [in this window] [in a new window]   Figure 1. Pressure–internal diameter (left panel) and stress–strain relationships (right panel) in cerebral arterioles during maximal dilatation with EDTA in untreated (n=5) and L-NAME–treated mice (n=7). Values are mean±SEM. *P<0.05 versus untreated. D indicates cerebral arteriolar diameter; D0, original cerebral arteriolar diameter.

eNOS-Deficient Mice
Systemic mean arterial pressure was significantly greater in male and female homozygous eNOS-deficient mice than in male and female wild-type mice in both the unanesthetized and anesthetized states, even though anesthesia significantly reduced systemic mean arterial pressure in all groups (Table 2). Unanesthetized and anesthetized systemic mean arterial pressures in male and female heterozygous eNOS-deficient mice were intermediate between male and female homozygous eNOS-deficient mice and male and female wild-type mice, but differences between the heterozygous eNOS-deficient groups and wild-type and homozygous eNOS groups were not significant (Table 2). Cerebral arteriolar systolic and pulse pressures, but not diastolic and mean pressures, were significantly increased in male and female homozygous eNOS-deficient mice, but not in male and female heterozygous eNOS-deficient mice (Table 2). Diameter before deactivation with EDTA was not significantly different in cerebral arterioles of male and female wild-type mice, male and female heterozygous eNOS-deficient mice, or male and female homozygous eNOS-deficient mice (Table 2). During deactivation with EDTA, internal and external diameters and cross-sectional area of the vessel wall were greater in cerebral arterioles in male and female homozygous eNOS-deficient mice than in male and female wild-type or heterozygous eNOS-deficient mice (Table 2). Thus, cerebral arterioles in homozygous, but not heterozygous, eNOS-deficient mice of both sexes underwent hypertrophy of the vessel wall.

View this table: [in this window] [in a new window]   Table 2. Baseline Values in eNOS-Deficient Male Mice

Effects of Carotid Ligation
Carotid ligation significantly reduced both mean and pulse pressures in cerebral arterioles in wild-type and homozygous eNOS-deficient mice (Figure 2). Mean pressure in ligated cerebral arterioles was significantly greater in homozygous eNOS-deficient mice than in wild-type mice, whereas pulse pressure ipsilateral to the ligation was not significantly different in the 2 groups. Thus, carotid ligation reduced pulse pressure more effectively than mean pressure in cerebral arterioles of homozygous eNOS-deficient mice.

View larger version (22K): [in this window] [in a new window]   Figure 2. Unanesthetized mean pressure (left panel), pulse pressure (middle panel), and cross-sectional area of vessel wall (CSA; right panel) in sham-operated (open bars) and ligated (solid bars) cerebral arterioles in wild-type mice (n=12) and homozygous eNOS-deficient mice (n=12). Values are mean±SEM. *P<0.05 versus sham-operated wild-type; P<0.05 versus sham-operated eNOS-deficient. WT indicates wild-type; HOMO, homozygous.

Cross-sectional area of the vessel wall was significantly less in ligated than in sham arterioles in wild-type mice (Figure 2). In contrast, cross-sectional was not significantly different in sham and ligated arterioles in homozygous eNOS-deficient mice (Figure 2). Thus, carotid ligation in homozygous eNOS-deficient mice did not prevent hypertrophy of cerebral arterioles, even though it normalized arteriolar pulse pressure.

Vascular Mechanics
Internal diameter in cerebral arterioles during maximal dilatation was larger in male and female homozygous eNOS-deficient mice, but not in male and female heterozygous eNOS-deficient, than in male and female wild-type mice at all levels of arteriolar pressure between 10 and 40 mm Hg (Figure 3). The stress–strain curves in cerebral arterioles in male and female homozygous and male and female heterozygous eNOS-deficient mice were similar to the curve in cerebral arterioles in male and female wild-type mice (Figure 4). In addition, the slope of tangential elastic modulus versus stress was not significantly different in male and female homozygous and male and female heterozygous eNOS-deficient mice and male and female wild-type mice (Table 2). These findings suggest that eNOS deficiency in mice was not accompanied by alterations in passive distensibility of cerebral arterioles.

View larger version (19K): [in this window] [in a new window]   Figure 3. Pressure–internal diameter relationships in maximally dilated cerebral arterioles in male (left panel) and female (right panel) wild-type (6 males and 4 females), heterozygous (6 males and 5 females), and homozygous mice (5 males and 5 females). Values are mean±SEM. *P<0.05 versus wild-type.

View larger version (19K): [in this window] [in a new window]   Figure 4. Stress–strain relationships in maximally dilated cerebral arterioles in male (left panel) and female (right panel) wild-type (6 males and 4 females), heterozygous (6 males and 5 females), and homozygous mice (5 males and 5 females). Values are mean±SEM. D indicates cerebral arteriolar diameter; D0, original cerebral arteriolar diameter.

	Discussion

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There were 3 major new findings in this study. First, chronic hypertension induced by treatment with L-NAME resulted in hypertrophy of cerebral arterioles of C57BL/6J mice. Second, hypertrophy of cerebral arterioles developed in mice with homozygous eNOS deficiency. Furthermore, unilateral carotid ligation in homozygous eNOS-deficient mice did not prevent hypertrophy in cerebral arterioles, and it normalized arteriolar pulse pressure, but not mean pressure. These findings suggest that eNOS deficiency promotes cerebral arteriolar hypertrophy independently of increases in arterial pulse pressure. Previous studies using models of chronic reduction in blood flow³⁴ and mechanical injury of the vessel wall³⁵ in eNOS-deficient mice have provided direct evidence that eNOS inhibits vascular growth. The findings in this study support this concept. Furthermore, by using an approach that does not alter cerebral blood flow or directly injure the cerebral arteriolar wall, this study provides direct evidence that eNOS inhibits vascular growth independently of blood flow or mechanical injury. Third, in contrast to homozygous eNOS deficiency, hypertrophy in cerebral arterioles did not develop in mice with heterozygous eNOS deficiency. This finding indicates that deletion of 1 copy of the eNOS gene is not sufficient to induce cerebral arteriolar hypertrophy. In addition, the finding may be relevant to humans with polymorphisms in the eNOS gene, which have been reported to alter the baseline production or bioavailability of NO.³⁶

Cerebral arterioles undergo hypertrophy in several models of experimental hypertension, including SHRSP, SHR, and renal hypertension.^3,33 Determinants that may contribute to cerebral vascular hypertrophy during chronic hypertension include increases in pressure,^4,37 neurohumoral factors,^8–11 genetic factors,^38,39 and endothelial factors, such as release of endothelin.¹²

Another endothelium-derived product that may contribute to regulation of vessel growth is NO. Consideration for this possibility first emerged with the finding that NO suppresses mitogenesis and proliferation of vascular muscle in tissue culture.¹³ However, studies in which NO synthase inhibitors were used to suppress production of NO in living animals have yielded conflicting results with respect to the importance of NO in vascular growth. Dunn and Gardiner⁴⁰ found that treatment with L-NAME produced no change in cross-sectional area of the vessel wall in mesenteric resistance arteries in Brattleboro rats. Although Schiffrin et al^41,42 found an increase in cross-sectional area of the vessel wall in mesenteric resistance arteries in L-NAME–treated rats, the increase was smaller in magnitude (cross-sectional area [CSA] of 7% to 10%) than that often found by other investigators in other models of hypertension that have levels of arterial pressure similar to those in rats treated with L-NAME, such as SHR⁴³ (CSA of 70%) and renal hypertensive rats⁴⁴ (CSA of 40%). In contrast, we found in a previous study¹⁴ that treatment of rats with L-NAME resulted in an increase in cross-sectional area of the vessel wall in cerebral arterioles, and the magnitude was greater (CSA of 50% to 80%) than in small mesenteric arteries^40–42 and was similar to, or greater than, increases found in cerebral arterioles in other models of hypertension that have similar increases in arterial pressure, including SHR (CSA of 58%),³ SHRSP (CSA of 30%),³³ and renal hypertensive rats (CSA of 43%).³

The present study confirms our previous findings in rats.¹⁴ Treatment with L-NAME resulted in hypertrophy of cerebral arterioles in mice. Furthermore, the magnitude of cerebral arteriolar hypertrophy induced in mice by L-NAME treatment (CSA of 40%) was similar to the magnitude induced in rats, even though increases in systemic arterial pressure were considerably less in L-NAME–treated mice (systemic arterial mean pressure [SAP] of 18%) than in L-NAME–treated rats (SAP of 65%). Thus, the findings in this study, together with our previous study,¹⁴ suggest that inhibition of NO synthase, with chronic hypertension, may produce substantial hypertrophy in the cerebral circulation. Furthermore, the findings also support the concept that NO may play an important role in the regulation of vascular growth.

At least 3 potential problems complicate interpretation of the effects of L-NAME treatment on vascular structure. First, one must consider the possibility that L-NAME promotes vascular hypertrophy through a mechanism unrelated to its effects on NOS inhibition. For example, L-NAME may stimulate hypertrophy directly⁴⁵ or indirectly through activation of angiotensin II AT₁ receptors.^22–24 The second potential problem is that treatment with L-NAME produces significant hypertension in rats and mice. One must consider, therefore, the possibility that hypertrophy of cerebral arterioles in L-NAME–treated animals results directly from increases in arteriolar pressure per se rather than a nonpressor effect of L-NAME. A third potential problem with L-NAME treatment is that NOS inhibitors, including L-NAME, nonselectively inhibit all isoforms of NOS. In addition to eNOS in endothelium, nNOS is expressed in vascular muscle.¹⁵ Furthermore, perivascular nerves and brain tissue contain large amounts of nNOS,^16,17 and nNOS has been shown to influence cerebral vascular tone^18,19 and may therefore also influence cerebral vascular growth. Thus, even if the other potential problems associated with L-NAME treatment can be eliminated, its nonspecificity means that no conclusions regarding effects of L-NAME on cerebral vascular growth can be drawn with respect to which of the NOS isoforms are involved.

We addressed the potential problems with L-NAME treatment in this study by examining cerebral arterioles in eNOS-deficient mice. Our finding that cerebral arterioles in homozygous eNOS-deficient mice undergo hypertrophy supports the concept that eNOS plays a major role in regulation of cerebral vascular growth. The findings also support the suggestion that hypertrophy induced with L-NAME treatment is related to its effects of NO synthase inhibition rather than its effects on arterial pressure or AT₁ receptors.

An important consideration in this study is whether hypertrophy of cerebral arterioles in homozygous eNOS-deficient mice resulted directly from increases in arteriolar pressure per se. To test this possibility, we examined effects of unilateral carotid ligation, which we previously found to normalize pulse pressure and prevent hypertrophy in cerebral arterioles of SHRSP.⁶ In this study, carotid ligation did not prevent hypertrophy of cerebral arterioles in homozygous eNOS-deficient mice, even though increases in arteriolar pulse pressure were prevented. However, carotid ligation failed to normalize mean pressure in cerebral arterioles. Thus, we cannot rule out the possibility that hypertrophy in cerebral arterioles of homozygous eNOS-deficient mice was the result of increases in mean pressure. There are 3 reasons to consider this possibility less likely, however. First, as demonstrated in this study, carotid ligation significantly reduced cross-sectional area of the arteriolar wall in wild-type mice, even though the reduction in arteriolar pulse pressure was less in wild-type mice (5 mm Hg) than in eNOS-deficient mice (11 mm Hg). Second, carotid ligation prevents cerebral arteriolar hypertrophy in SHRSP, even though ligation only partially attenuates increases in arteriolar systolic and mean pressures.⁶ Finally, arteriovenous fistulae in rats increases pulse pressure, but not mean pressure, in cerebral arterioles, and results in hypertrophy of the arteriolar wall.⁴⁶

We were surprised in this study to find that cerebral arteriolar hypertrophy did not develop in heterozygous eNOS mice. We found previously that responses of carotid artery to acetylcholine, nitroprusside, and serotonin are altered in heterozygous eNOS-deficient mice, although to a lesser degree than in homozygous eNOS-deficient mice.^25,26 Based on this finding, we anticipated that some degree of cerebral arteriolar hypertrophy might develop in heterozygous, as well as homozygous, eNOS-deficient mice, although possibly to a lesser extent than in the homozygous group. Cross-sectional area of the vessel wall, however, was virtually the same in cerebral arterioles of heterozygous eNOS-deficient mice and wild-type mice. Thus, in contrast to the apparent "dosing" effect of the eNOS gene on some vascular responses,^25,26 deletion of a single copy of the eNOS gene is not sufficient to induce cerebral arteriolar hypertrophy.

The mechanism by which NO formed by eNOS may contribute to cerebral vascular growth and hypertrophy is unclear and may involve a number of possibilities. One possibility is that NO affects vascular growth through regulation of endothelin-1 gene expression in the vessel wall.⁴⁷ This suggestion is based on the finding that treatment with L-NAME results in increases in severity of hypertrophy in aorta and large mesenteric arteries in SHRSP together with increases in endothelin-1 mRNA in the vessel wall.⁴⁷ In contrast, neither severity of hypertrophy nor endothelin-1 mRNA is increased in small mesenteric resistance arteries of SHRSP during treatment with L-NAME.⁴⁸ These findings suggest that L-NAME stimulates production of endothelin-1 in large, but not small, arteries, and thus results in additional hypertrophy only in large arteries as a consequence of the trophic effects of endothelin-1.^49,50 Based on this assumption, one might conclude that in this study, endothelin-1 did not contribute to hypertrophy of cerebral arterioles in L-NAME–treated rats. However, we are unaware of any evidence to indicate that L-NAME does not increase endothelin-1 mRNA in cerebral arterioles. Thus, one cannot rule out a role for endothelin-1 in the development of cerebral arteriolar hypertrophy during treatment with L-NAME.

Another possible mechanism linking NO to vascular growth involves platelet-derived growth factor (PDGF). PDGF is a powerful growth factor for vascular muscle.^51,52 PDGF apparently exerts its growth effects in part through the protein kinase B cascade.⁵³ Recent evidence suggests that NO selectively attenuates PDGF-induced increases in protein kinase B- activation, which in turn may contribute to diminished proliferation of vascular muscle.⁵³ Thus, NO may contribute to inhibition of vascular growth by inactivating PDGF-induced growth of vascular muscle. Correspondingly, reduced availability of NO may contribute to vascular hypertrophy by increased PDGF activity.

	Acknowledgments

 
We thank Tom Gerhold and Shams Ghomeim for technical assistance.

This work was supported by National Institutes of Health grants HL-22149, NS-24621, HL-38901, and HL-62984.

	Footnotes

 
Original received October 22, 2001; resubmission received July 8, 2004; revised resubmission received August 25, 2004; accepted September 15, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Heagerty AM, Aalkjaer C, Bund SJ, Korsgaard N, Mulvany MJ. Small artery structure in hypertension: Dual processes of remodeling and growth. Hypertension. 1993; 21: 391–397.[Free Full Text]

2. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic hypertension. Hypertension. 1989; 13: 968–972.[Abstract/Free Full Text]

3. Baumbach GL, Hajdu MA. Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats. Hypertension. 1993; 21: 816–826.[Abstract/Free Full Text]

4. Folkow B, Gurevich M, Hallbäck M, Lundgren Y, Weiss L. The hemodynamic consequences of regional hypotension in spontaneously hypertensive and normotensive rats. Acta Physiol Scand. 1971; 83: 532–541.[Medline] [Order article via Infotrieve]

5. Hajdu MA, Heistad DD, Baumbach GL. Effects of antihypertensive therapy on mechanics of cerebral arterioles in rats. Hypertension. 1991; 17: 308–316.[Abstract/Free Full Text]

6. Baumbach GL, Siems JE, Heistad DD. Effects of local reduction in pressure on distensibility and composition of cerebral arterioles. Circ Res. 1991; 68: 338–351.[Abstract/Free Full Text]

7. Christensen KL. Reducing pulse pressure in hypertension may normalize small artery structure. Hypertension. 1991; 18: 722–727.[Abstract/Free Full Text]

8. Baumbach GL, Heistad DD, Siems JE. Effect of sympathetic nerves on composition and distensibility of cerebral arterioles in rats. J Physiol (Lond). 1989; 416: 123–140.[Abstract/Free Full Text]

9. Lee RMKW, Triggle CR, Cheung D, Coughlin M. Structural and functional consequences of neonatal sympathectomy on the blood vessels of spontaneously hypertensive rats. Hypertension. 1987; 10: 328–338.[Abstract/Free Full Text]

10. Fischli W, Hefti F, Clozel J-P. Effects of acute and chronic cilazapril treatment in spontaneously hypertensive rats. Br J Clin Pharmacol. 1989; 27: 151S–158S.[Medline] [Order article via Infotrieve]

11. Geisterfer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988; 62: 749–756.[Abstract/Free Full Text]

12. Chillon JM, Heistad DD, Baumbach GL. Effects of endothelin receptor inhibition on cerebral arterioles in hypertensive rats. Hypertension. 1996; 27: 794–798.[Abstract/Free Full Text]

13. 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.[Medline] [Order article via Infotrieve]

14. Chillon JM, Ghoneim S, Baumbach GL. Effects of chronic nitric oxide synthase inhibition on cerebral arterioles in rats. Hypertension. 1997; 30: 1097–1104.[Abstract/Free Full Text]

15. Brophy CM, Knoepp L, Xin J, Pollock JS. Functional expression of NOS 1 in vascular smooth muscle. Am J Physiol. 2000; 278: H991–H997.

16. Yang G, Iadecola C. Activation of cerebellar climbing fibers increases cerebellar blood flow: role of glutamate receptors, nitric oxide, and cGMP. Stroke. 1998; 29: 499–508.[Abstract/Free Full Text]

17. Iadecola C. Regulation of the cerebral microcirculation during neural activity: is nitric oxide the missing link? Trends Neurosci. 1993; 16: 206–214.[CrossRef][Medline] [Order article via Infotrieve]

18. Yang G, Chen G, Ebner TJ, Iadecola C. Nitric oxide is the predominant mediator of cerebellar hyperemia during somatosensory activation in rats. Am J Physiol. 1999; 277: R1760–R1770.[Medline] [Order article via Infotrieve]

19. Faraci FM, Breese KR. Nitric oxide mediates vasodilatation in response to activation of N-methyl-D-aspartate receptors in brain. Circ Res. 1993; 72: 476–480.[Abstract/Free Full Text]

20. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA. Hypertension in mice lacking the gene for endothelial nitric oxide synthase [see comments]. Nature. 1995; 377: 239–242.[CrossRef][Medline] [Order article via Infotrieve]

21. Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, Smithies O. Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1996; 93: 13176–13181.[Abstract/Free Full Text]

22. Pollock DM, Polakowski JS, Divish BJ, Opgenorth TJ. Angiotensin blockade reverses hypertension during long-term nitric oxide synthase inhibition. Hypertension. 1993; 21: 660–666.[Abstract/Free Full Text]

23. Jover B, Herizi A, Ventre F, Dupont M, Mimran A. Sodium and angiotensin in hypertension induced by long-term nitric oxide blockade. Hypertension. 1993; 21: 944–948.[Abstract/Free Full Text]

24. Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension. 1992; 20: 298–303.[Abstract/Free Full Text]

25. Faraci FM, Sigmund CD, Shesely EG, Maeda N, Heistad DD. Responses of carotid artery in mice deficient in expression of the gene for endothelial NO synthase. Am J Physiol. 1998; 274: H564–H570.[Medline] [Order article via Infotrieve]

26. Lamping KG, Faraci FM. Role of sex differences and effects of endothelial NO synthase deficiency in responses of carotid arteries to serotonin. Arterioscler Thromb Vasc Biol. 2001; 21: 523–528.[Abstract/Free Full Text]

27. Takahashi N, Smithies O. Human genetics, animal models and computer simulations for studying hypertension. Trends Genet. 2004; 20: 136–145.[CrossRef][Medline] [Order article via Infotrieve]

28. Davisson RL, Kim HS, Krege JH, Lager DJ, Smithies O, Sigmund CD. Complementation of reduced survival, hypotension, and renal abnormalities in angiotensinogen-deficient mice by the human renin and human angiotensinogen genes. J Clin Invest. 1997; 99: 1258–1264.[Medline] [Order article via Infotrieve]

29. Lake-Bruse KD, Faraci FM, Shesely EG, Maeda N, Sigmund CD, Heistad DD. Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice. Am J Physiol. 1999; 277: H770–H776.[Medline] [Order article via Infotrieve]

30. Baumbach GL, Sigmund CD, Bottiglieri T, Lentz SR. Structure of cerebral arterioles in cystathionine beta-synthase-deficient mice. Circ Res. 2002; 91: 931–937.[Abstract/Free Full Text]

31. Baumbach GL, Sigmund CD, Faraci FM. Cerebral arteriolar structure in mice overexpressing human renin and angiotensinogen. Hypertension. 2003; 41: 50–55.[Abstract/Free Full Text]

32. Harper SL, Bohlen HG. Microvascular adaptation in the cerebral cortex of adult spontaneously hypertensive rats. Hypertension. 1984; 6: 408–419.[Abstract/Free Full Text]

33. Baumbach GL, Dobrin PB, Hart MN, Heistad DD. Mechanics of cerebral arterioles in hypertensive rats. Circ Res. 1988; 62: 56–64.[Abstract/Free Full Text]

34. Rudic RD, Shesely EG, Maeda N, Smithies O, Segal SS, Sessa WC. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest. 1998; 101: 731–736.[Medline] [Order article via Infotrieve]

35. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL. Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice. J Clin Invest. 1998; 101: 1225–1232.[Medline] [Order article via Infotrieve]

36. Veldman BA, Spiering W, Doevendans PA, Vervoort G, Kroon AA, de Leeuw PW, Smits P. The Glu298Asp polymorphism of the NOS 3 gene as a determinant of the baseline production of nitric oxide. J Hypertens. 2002; 20: 2023–2027.[CrossRef][Medline] [Order article via Infotrieve]

37. Weiss L, Hallbäck M. Time course and extent of structural vascular adaptation to regional hypotension in adult spontaneously hypertensive rats (SHR). Acta Physiol Scand. 1974; 91: 365–373.[Medline] [Order article via Infotrieve]

38. Lee RMKW. Vascular changes at the prehypertensive phase in the mesenteric arteries from spontaneously hypertensive rats. Blood Vessels. 1985; 22: 105–126.[Medline] [Order article via Infotrieve]

39. Eccleston-Joyner CA, Gray SD. Arterial hypertrophy in the fetal and neonatal spontaneously hypertensive rat. Hypertension. 1988; 12: 513–518.[Abstract/Free Full Text]

40. Dunn WR, Gardiner SM. No evidence for vascular remodelling during hypertension induced by chronic inhibition of nitric oxide synthase in Brattleboro rats. J Hypertens. 1995; 13: 849–857.[CrossRef][Medline] [Order article via Infotrieve]

41. Li J-S, Schiffrin EL. Resistance artery structure and neuroeffector mechanisms in hypertension induced by inhibition of nitric oxide synthase. Am J Hypertens. 1994; 7: 996–1004.[Medline] [Order article via Infotrieve]

42. Deng LY, Thibault G, Schiffrin EL. Effect of hypertension induced by nitric oxide synthase inhibition on structure and function of resistance arteries in the rat. Clin Exper Hypertens. 1993; 15: 527–537.[CrossRef]

43. Lee RMKW, Garfield RE, Forrest JB, Daniel EE. Morphometric study of structural changes in the mesenteric blood vessels of spontaneously hypertensive rats. Blood Vessels. 1983; 20: 57–71.[Medline] [Order article via Infotrieve]

44. Korsgaard N, Mulvany MJ. Cellular hypertrophy in mesenteric resistance vessels from renal hypertensive rats. Hypertension. 1988; 12: 162–167.[Abstract/Free Full Text]

45. Schiffrin EL. Vascular structure in N^G-nitro-L-arginine methyl ester-induced hypertension: methodological considerations for studies of small arteries in hypertension. J Hypertens. 1995; 13: 817–821.[CrossRef][Medline] [Order article via Infotrieve]

46. Baumbach GL. Effects of increased pulse pressure on cerebral arterioles. Hypertension. 1996; 27: 159–167.[Abstract/Free Full Text]

47. Sventek P, Li JS, Grove K, Deschepper CF, Schiffrin EL. Vascular structure and expression of endothelin-1 gene in L-NAME-treated spontaneously hypertensive rats. Hypertension. 1996; 27: 49–55.[Abstract/Free Full Text]

48. Li JS, Deng LY, Grove K, Deschepper CF, Schiffrin EL. Comparison of effect of endothelin antagonism and angiotensin-converting enzyme inhibition on blood pressure and vascular structure in spontaneously hypertensive rats treated with Nomega-nitro-L-arginine methyl ester - Correlation with topography of vascular endothelin-1 gene expression. Hypertension. 1996; 28: 188–195.[Abstract/Free Full Text]

49. Bobik A, Grooms A, Millar JA, Mitchell A, Grinpukel S. Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol. 1990; 258: C408–C415.[Medline] [Order article via Infotrieve]

50. Hirata Y, Takagi Y, Fukuda Y, Marumo F. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis. 1989; 78: 225–228.[CrossRef][Medline] [Order article via Infotrieve]

51. Cornwell TL, Arnold E, Boerth NJ, Lincoln TM. Inhibition of smooth muscle cell growth by nitric oxide and activation of cAMP-dependent protein kinase by cGMP. Am J Physiol. 1994; 267: C1405–C1413.[Medline] [Order article via Infotrieve]

52. Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science. 1991; 253: 1129–1132.[Abstract/Free Full Text]

53. Sandirasegarane L, Charles R, Bourbon N, Kester M. NO regulates PDGF-induced activation of PKB but not ERK in A7r5 cells: implications for vascular growth arrest. Am J Physiol. 2000; 279: C225–C235.

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