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Integrative Physiology |
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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Key Words: hypertension L-NAME endothelial nitric oxide synthase deficiency vascular hypertrophy
| Introduction |
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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 tone18,19 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 eNOS20,21 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-NAMEtreated 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.2224
| Materials and Methods |
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L-NAMETreated 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-NAMEtreated 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-NAMEtreated 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 g1 body weight intraperitoneally), intubated, and mechanically ventilated with room air and supplemental O2. Additional anesthesia (1.7 mg 100 g1 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 Pressurediameter 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 pressurediameter 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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Vascular Mechanics
Internal diameter in cerebral arterioles during maximal dilatation was larger in L-NAMEtreated mice than in untreated mice at all levels of arteriolar pressure between 10 and 40 mm Hg (Figure 1, left panel). The stressstrain curve in cerebral arterioles in L-NAMEtreated 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-NAMEtreated mice and untreated mice (Table 1). These findings suggest that L-NAMEinduced hypertension was not accompanied by alterations in passive distensibility of cerebral arterioles, despite hypertrophy of the vessel wall.
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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.
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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.
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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 stressstrain 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.
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| Discussion |
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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,811 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 al41,42 found an increase in cross-sectional area of the vessel wall in mesenteric resistance arteries in L-NAMEtreated 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 arteries4042 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-NAMEtreated mice (
systemic arterial mean pressure [SAP] of
18%) than in L-NAMEtreated 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.2224 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-NAMEtreated 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 tone18,19 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-NAMEtreated 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 |
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This work was supported by National Institutes of Health grants HL-22149, NS-24621, HL-38901, and HL-62984.
| Footnotes |
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| References |
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