(Hypertension. 2000;35:1055.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Vascular NADH/NADPH Oxidase Is Involved in Enhanced Superoxide Production in Spontaneously Hypertensive Rats
From the Vascular Pathophysiology Unit, School of Medicine, University of Navarra, Pamplona, Spain.
Correspondence to Javier Díez, MD, PhD, Unidad de Fisiopatología Vascular, Facultad de Medicina, C/Irunlarrea s/n, 31080 Pamplona, Spain. E-mail jadimar{at}unav.es
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Abstract—This study was designed to test the hypothesis that stimulation of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase is involved in increased vascular superoxide anion (·O2-) production in spontaneously hypertensive rats (SHR). The study was performed in 16-week-old and 30-week-old normotensive Wistar-Kyoto rats (WKY16 and WKY30, respectively) and in 16-week-old and 30-week-old SHR (SHR16 and SHR30, respectively). In addition, 16-week-old SHR were treated with oral irbesartan (average dose 20 mg/kg per day) for 14 weeks (SHR30-I). Aortic NADH/NADPH oxidase activity was determined by use of chemiluminescence with lucigenin. The expression of p22phox messenger RNA was assessed by competitive reverse transcription–polymerase chain reaction. Vascular responses to acetylcholine were determined by isometric tension studies. Aortic wall structure was studied, determining the media thickness and the cross-sectional area by morphometric analysis. Whereas systolic blood pressure was significantly increased in the 2 groups of hypertensive animals compared with their normotensive controls, no differences were observed in systolic blood pressure between SHR30 and SHR16. No other differences in the parameters measured were found between WKY16 and SHR16. In SHR30 compared with WKY30, we found significantly greater p22phox mRNA level, NADH/NADPH-driven ·O2- production, media thickness, and cross-sectional area and an impaired vasodilation in response to acetylcholine. Treated SHR had similar NADH/NADPH oxidase activity and p22phox expression as the WKY30 group. The vascular functional and morphological parameters were improved in SHR30-I. These findings suggest that an association exists between p22phox gene overexpression and NADH/NADPH overactivity in the aortas of adult SHR. Enhanced NADH/NADPH oxidase–dependent ·O2- production may contribute to endothelial dysfunction and vascular hypertrophy in this genetic model of hypertension.
Key Words: endothelium • rats, inbred SHR • superoxides • hypertrophy
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Introduction |
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An exaggerated production of superoxide anion ( ·O2-) by the vascular wall has been observed in different animal models of hypertension, including spontaneously hypertensive rats (SHR).1 2 3 In the majority of cases, the source of ·O2- is uncertain, although involvement of endothelial nitric oxide synthase4 5 and xanthine oxidase6 7 have been suggested. A growing amount of evidence supports the possibility that increased oxidative inactivation of nitric oxide (NO) by an excess of ·O2- may account for the decrease in available NO and endothelial dysfunction seen in SHR.8 9 10 On the other hand, recent findings suggest that ·O2- may participate in vascular hypertrophy by stimulating the growth of vascular smooth muscle cells (VSMCs).11 12
Several observations suggest that the nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase system accounts for the majority of ·O2- generation in the vessel wall.13 14 The vascular NADH/NADPH oxidase appears to be structurally and functionally different from the neutrophil oxidase. The endothelial and VSMC NADH/NADPH oxidases are probably not all similar. All components of the NADPH-oxidase enzyme have been found in endothelial cells.15 In contrast, only p22phox could be identified in VSMCs and has been shown to participate in the increased ·O2- production on stimulation with angiotensin II.16
We have hypothesized that stimulation of NADH/NADPH oxidase may contribute to increased vascular ·O2- production in SHR. Thus, the present study was designed to test whether NADH/NADPH oxidase activity is enhanced and p22phox gene expression is upregulated in the aortas of SHR. In addition, we also investigated whether blockade of angiotensin II type 1 (AT1) receptors with irbesartan modifies the enzyme in SHR.
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Methods |
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Animals and Design
The investigation was performed according to the European Community guidelines for animal ethical care and use of laboratory animals (Directive 86/609). Experiments were performed with rats provided by Harlan UK Limited (Blackthorn, Bicester, Oxon, UK). The rats were housed in cages maintained in a humidity- and temperature-controlled room and fed a standard diet. Sixteen-week-old male SHR (n=90) and their normotensive genetic controls, Wistar-Kyoto rats (WKY) (n=60), were studied in the following manner: (1) 16-week-old WKY (n=30) and SHR (n=30) were killed to perform studies in young rats (groups WKY16 and SHR16, respectively); (2) 16-week-old WKY (n=30) and SHR (n=30) were observed in our colony for additional 14 weeks and killed at 30 weeks of age to perform studies in adult rats (groups WKY30 and SHR30, respectively), and (3) 16-week-old SHR (n=30) were treated with irbesartan and killed at 30 weeks. The drug was dissolved in drinking water and the concentration was adjusted for the daily water intake and body weight to obtain an average daily dose of 20 mg/kg body wt. In each group of animals, 10 rats were used to measure endothelial function in intact vessels and NADH/NADPH oxidase activity in vessel homogenates, 10 rats were used to determine vascular p22phox messenger RNA (mRNA) expression, and the remaining 10 rats were used for aortic morphometric analysis.
Systolic blood pressure (SBP) was measured every 2 weeks by the standard tail-cuff method. For measurement of isometric wall tension and enzymatic and molecular studies, rats were killed by decapitation. The thoracic aorta then was carefully excised and dissected, adventitial tissues were carefully removed, and the vascular wall was washed thoroughly with normal saline to remove any contaminating blood.
Measurement of ·O2- Production
in Aortic Homogenates
Vascular ·O2- was
estimated with lucigenin-enhanced chemiluminescence.17
Aortic segments (2 to 3 cm) were placed in chilled modified Krebs/HEPES
buffer and homogenized on ice with a glass/glass
motor-driven tissue homogenizer for 2 minutes in
50 mmol/L PBS, which contained 0.01 mmol/L EDTA. In some
experiments the endothelium was removed by rubbing. The
homogenate was centrifuged at 1000g for
10 minutes. The pellet was discarded and the supernatant was stored on
ice until use. Protein content was measured by the method of
Bradford.18 NADH or NADPH oxidase activity was
measured by chemiluminescence in a scintillation vial containing HEPES
buffer, 250 µmol/L lucigenin, in 2 mL buffer phosphate. The
chemiluminescence, which occurred over the ensuing 5 minutes in
response to the addition of either 100 µmol/L NADH or 100
µmol/L NADPH, was recorded. No activity could be measured in the
absence of NADH or NADPH. Reactions were initiated by the addition of
20 µL homogenate. To determine the
·O2- dependency of the
lucigenin-enhanced chemiluminescence obtained from vascular
homogenates, we examined the effect of
Cu2+/Zn2+ superoxide
dismutase (SOD) (2000 U/mL) on
·O2- production after
stimulation of homogenates with NADH. In some experiments,
membranes and cytosol were separated by centrifugation
(50 000g for 30 minutes). Twenty-five microliters of either
the supernatant or the particulate fraction were used to examine NADH
oxidase–dependent and NADPH oxidase–dependent
·O2- production of
these subcellular fractions.
Endothelium-Dependent and
Endothelium-Independent Relaxations
After washing, each aorta was cut into 2- to 3-mm rings (6 per
rat). The rings were suspended in a vessel that contained 10 mL of
Krebs-Henseleit solution with the following composition (in mmol/L):
118.4 NaCl, 4.74 KCl, 1.18 MgSO2, 0.7
H2O, 1.19
KH2PO4, 2.52
CaCl2, 0.2 H2O, 25
NaHCO3, and 11.5 glucose (pH, 7.34). The solution
was oxygenated with a 95% O2–5%
CO2 mixture and maintained at 37°C in a
thermostated bath. Two stainless steel wires were inserted into the
vascular lumen; one was anchored to a stationary support and the other
was connected to a force isometric transducer. The rings were incubated
for 60 minutes for equilibration at a resting tension of 2g
with buffer exchanges every 15 minutes during this period. Changes in
isometric forces were analyzed and recorded by an Isolated
Organs Data Acquisition Program (Proto5). The rings were then maximally
contracted with norepinephrine (10-7
mol/L), and once a plateau had been reached, the
endothelium-dependent and
endothelium-independent relaxations were studied by
plotting the concentration-response curves for the responses to
10-9 to 10-5 mol/L
acetylcholine (Ach) and to 10-9 to
10-6 mol/L sodium nitroprusside (SNP),
respectively. Endothelium-dependent and
endothelium-independent relaxations were expressed as
percentages of decline of the contraction induced by
norepinephrine. These values were then plotted against the
negative logarithm of the agonist dose to produce dose-response curves.
Dose-response curves were analyzed, and the
EC50 value (the concentration for half-maximal
response) for relaxation of each ring was calculated by logistic
analysis and expressed as the pD2 defined
as -log (EC50).
Reverse Transcription–Polymerase Chain Reaction for
p22phox
Total RNA was extracted from homogenized thoracic
aortas with the use of Ultraspec RNA reagent. mRNA was isolated from
total RNA with the Oligotex mRNA kit (Qiagen), and mRNA concentration
was determined spectrometrically. The expression of p22phox
was quantified in the aorta by a competitive reverse
transcription–polymerase chain reaction (RT-PCR) method, as described
by Gilliland et al.19 On the basis of rat
p22phox cDNA sequence,20 the
oligonucleotides 5'-GCTCATCTGTCTGCTGGAGTA-3' and
5'-ACGACCTCATCTGTCACTGGA-3' were selected as sense and antisense
primers, respectively. The p22phox-specific primers
amplified a 435-bp PCR product. The identity of this PCR
product was verified by sequencing. The heterologous DNA template
(p22phox competitor) was constructed with the use of a
PCR-MIMIC construction kit (Clontech), following the manufacturer’s
instructions. The obtained 318-bp p22phox competitor
contained the p22phox gene–specific sequences incorporated
at the ends. This 318-bp fragment was cloned in a pGEMT vector
(Promega), linearized by digestion with Sca I, and subjected
to in vitro transcription into RNA. The purified
p22phox-construct RNA was quantified
spectrometrically.
Both RT and PCR were performed in a single tube with gene-specific primers and specific target RNAs, with the use of the Superscript 1-step RT-PCR System (Life Technologies). RT-PCR was performed with constant amounts of aortic mRNA (35 ng) together with a range of amounts between 106 and 105 molecules of competitor RNA. RT-PCR reaction mixture (50 µL) contained aortic and competitor RNA, a buffer containing 0.2 mmol/L of each dNTP, and 1.2 mmol/L MgSO4, 20 pmol/L of primer, 20 U of Rnase inhibitor (Pharmacia Biotech Inc), and 1 µL of a RT/Taq Mix containing a mixture of Superscript II Reverse Transcriptase and Taq DNA Polymerase. All components necessary for RT-PCR were mixed in 1 tube, and RT (55°C for 30 minutes and then to 94°C for 2 minutes) was automatically followed by PCR cycling (35 cycles with a temperature profile of 94°C for 20 seconds, 60°C for 25 seconds, and 72°C for 1 minute and finally 5 minutes at 72°C for quantification of p22phox mRNA) without additional steps. The PCR products were run on a 2% agarose gel, stained with ethidium bromide, visualized by UV illumination, and photographed. The amounts of target and competitor PCR product were quantified by densitometric scanning of the film negative. To correct for differences in size of target (435 bp) and competitor (318 bp) p22phox PCR products, the amounts of competitor PCR product were multiplied by 1.368 (435/318). The ratio of target to competitor PCR product was plotted against the number of competitor construct molecules on a log-log scale. At the point in which equal molar amounts of target and competitor product were yielded by PCR (ie, competition ratio=1), the original number of transcript in the aortic sample was defined by the known input of competitor construct molecules.
Vascular Morphology
Before the animals were killed, they were weighted and
anesthetized (sodium thiopental, 30 mg/kg IP). Subsequently,
the thoracic aorta was fixed by retrograde perfusion as we have
previously described.21 In brief, the aorta was cannulated
with a polyethylene catheter, the heart was arrested in
diastole by an intracarotid injection of KCl (1.0 mol/L, 1
mL), and the right atria were incised to allow drainage of blood and
perfusate. After initial perfusion with normal saline buffer to
wash the blood, 4% paraformaldehyde was perfused for 5
minutes at a pressure equal to the mean arterial pressure
determined before the animals were killed. The thoracic aorta was
excised and transversally cut in 8 slices. The slices were
alternatively separated into 2 groups, postfixed for 5 hours by
immersion in buffered 4% paraformaldehyde and 10%
formalin, respectively, and finally, dehydrated and embedded in
paraffin. Transversal sections (4 µm in thickness) of the
thoracic aorta were obtained from the 4
paraformaldehyde-fixed, paraffin-embedded specimens.
Media thickness (MT) and cross-sectional area (CSA) were determined by
morphometry with an automated image analysis system (Visilog
4.1.5, Noesis) as previously described by van Gorp et
al.22 For this purpose, 1 section of each specimen (4 from
each rat) was stained for elastin fibers. Sections were
analyzed under the microscope (x4), and images of the whole
aortic ring were digitized (final resolution of 85.5
mm2/pixel). Stained elastic lamellae were
segmented by interactive gray-level thresholding, and after binary
processing and analysis, CSA and MT were obtained. CSA reflects
the degree of arterial hypertrophy better than
MT because it is not influenced by the variations in the perfusion
pressure.23
A monoclonal mouse antibody against rat monocyte/macrophages (ED1, Serotec)24 at a concentration of 1:100 in PBS was applied for 20 minutes. In control experiments, we checked that ED1 is specific for rat monocyte macrophages and does not recognize granulocytes, lymphocytes, endothelial cells, or smooth muscle cells. Fluorescein-conjugated goat anti-mouse antibody (Dakopatts) was used as a second antibody at a concentration of 1:40.
Statistical Analysis
Data are expressed as mean±SEM. Differences among the 5 groups
of rats were tested by a 1-way ANOVA. A Scheffé’s post hoc test
was used to examine differences between groups when significance was
indicated. Probability values <0.05 were considered significant.
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Blood Pressure
At the age of 16 weeks, SBP was higher (P<0.01) in SHR16 than in WKY16 (Table). Values of SBP remained higher in SHR than in WKY along the experimental 14-week period. Therefore, at the end of the 14-week-period of observation, SBP was higher (P<0.01) in SHR30 than in WKY30 (Table). No differences were found in SBP between SHR30 and SHR16. SBP was lower (P<0.01) in SHR30-I than in SHR30 (Table). Most rats (80%) in the SHR30-I group exhibited final values of SBP above the upper normal limit measured in WKY30 (176 mm Hg).
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p22phox mRNA Expression
In preliminary experiments, we proved that there were no
differences in the rate of transcription of the target and competitor
sequences. A representative competitive RT-PCR
experiment of aortic p22phox mRNA in young and adult rats is
shown in Figure 1a. In initial
experiments, p22phox was amplified from cDNA in the presence
of several serial dilutions of the p22phox competitor. The
results of these experiments enabled us to find the most appropriate
amounts of the p22phox competitor. Thus, definitive
experiments were performed with different amounts of p22phox
competitor comprised between 105 and
106 molecules. No differences in the
p22phox mRNA level were found between
WKY16 and SHR16 (3685±813
versus 4218±707 molecules of p22phox mRNA/ng mRNA) (Figure 1b). In contrast, p22phox mRNA level was 2.5-fold
higher in SHR30 than in
WKY30 (10 455±1056 versus 4221±349 molecules
of p22phox mRNA/ng mRNA, P<0.01) (Figure 1b). Furthermore, the p22phox mRNA level was greater
(P<0.01) in SHR30 than in
SHR16. After treatment with irbesartan,
p22phox mRNA expression was lower (P<0.01) in
SHR30-I (3456 molecules of p22phox
mRNA/ng mRNA) than in SHR30, and the values in
treated SHR were similar to those measured in
WKY30 (Figure 1b).
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NADH/NADPH Oxidase Activity
No differences were found between WKY16 and
SHR16 in NADH oxidase activity (5.45±0.66 versus
5.96±0.77 nmol
·O2- ·min-1 ·mg-1) and NADPH oxidase activity
(1.92±0.16 versus 1.73±0.27 nmol
·O2- ·min-1 ·mg-1) (Figure 2). In contrast, both activities were
higher (P<0.01) in SHR30 than in
WKY30 (11.01±0.81 versus 5.81±0.74 nmol
·O2- ·min-1 ·mg-1 for NADH oxidase activity; 5.09±0.41
versus 2.21±0.19 nmol
·O2- ·min-1 ·mg-1 for NADPH oxidase activity). The
activity of the oxidase was lower (P<0.01) in
SHR30-I than in SHR30
(5.50±0.53 nmol
·O2- ·min-1 ·mg-1 for NADH oxidase activity and
2.31±0.27 nmol
·O2- ·min-1 ·mg-1 for NADPH oxidase activity) (Figure 2).
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To assess the cellular source of increased NADH/NADPH oxidase activity in the vascular wall, some experiments were performed in deendothelized preparations. Although removal of the endothelium diminished slightly the NADH oxidase activity in aortic homogenates from SHR30 (with endothelium, 10.57±0.17 nmol ·O2- ·min-1 ·mg-1; without endothelium, 8.62±0.51 nmol ·O2- ·min-1 ·mg-1), the difference was not statistically significant.
To determine the ·O2- dependency of the lucigenin-enhanced chemiluminescence obtained from homogenates of rat aorta, we examined the effect of native Cu2+/Zn2+ SOD. As shown in Figure 3, SOD at very high concentrations (2000 U/mL) was effective in reducing the chemiluminescence signal in response to stimulation with NADH.
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NADH-dependent and NADPH-dependent oxidase activities in adult rats were located predominantly in the particulate fraction (Figure 4). NADH-driven ·O2- production measured in the particulate fraction was higher (P<0.01) in SHR30 than in WKY30 (159±16 versus 59±6 nmol · min-1 ·mg-1). NADPH-driven ·O2- production measured in the particulate fraction was also higher (P<0.01) in SHR30 than in WKY30 (48±3.7 versus 11.9±2.1 nmol · min-1 ·mg-1). Cytosolic activity was minimal in all the animals tested (Figure 4).
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High concentrations of lucigenin can produce a redox cycle with flavin-containing enzymes, leading to artifactual increases in estimates of ·O2-.25 This does not occur with low concentrations of lucigenin (5 µmol/L).26 Experiments with 5 µmol/L lucigenin revealed that NADH oxidase activity was greater (P<0.01) in aortas from SHR30 than in aortas from WKY30 (99±3 vs 56±3 counts · min-1 ·µg-1).
Vascular Relaxations
Whereas no differences in the maximal relaxations in response to
Ach were observed between WKY16 and
SHR16, this parameter was lower
(P<0.05) in SHR30 than in
WKY30 (Table). Furthermore, the response
to Ach was lower (P<0.05) in SHR30
than in SHR16 (Table). Irbesartan-treated
SHR exhibited a normal response to Ach (Table). There were no
significant differences in the sensitivity to Ach
(pD2 values) among the different groups of
animals (Table).
Maximum endothelium-independent relaxations to SNP were reached at the same concentration (3x10-6 mol/L) in hypertensive and normotensive rats, irrespective of age and treatment. There were no significant differences in the pD2 values between WKY16 and SHR16 (Table). Nevertheless, pD2 values were lower (P<0.05) in SHR30 and SHR30-I than in WKY30 (Table).
Morphological Analysis
There were no differences in MT and CSA between
WKY16 and SHR16
(Table). Otherwise, MT and CSA were greater (P<0.05)
in SHR30 than in WKY30 and
SHR16 (Table). Although the values of MT
and CSA were lower in SHR30-I than in
SHR30, the difference was not significant
(Table). No differences were observed in these 2
parameters between SHR30-I and
WKY30 (Table).
In WKY and SHR, immunofluorescence did not reveal any ED1-positive cells in the aorta. The absence of monocyte-macrophages in SHR aorta was confirmed by light microscopy and morphological evaluation.
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The observations of the present study provide the first indication that NADH/NADPH oxidase activity is increased in the aortic wall of adult SHR and that this overactivity is associated with upregulation of p22phox mRNA. In addition, we found that overactivity of NADH/NADPH oxidase was associated with impaired NO-dependent relaxation and media hypertrophy in the aortas of adult SHR. Interestingly, the enzyme abnormalities were not observed in the aortas of SHR chronically treated with the AT1 receptor antagonist irbesartan.
The NADH/NADPH oxidase is expressed in endothelial cells,12 VSMCs,12 16 27 adventitial fibroblasts,14 28 and inflammatory neutrophils.29 Although we did not perform in situ hybridization to determine which cell type(s) overexpress p22phox mRNA, several findings support a role for VSMCs as the potential source for oxidase overactivity found in adult SHR. First, the examination of NADH/NADPH activity was performed in aortas in which the adventitial tissue had been previously removed. Second, deendothelialization did not affect the level of NADH/NADPH oxidase activity. Third, increased NADH/NADPH oxidase activity was associated with hypertrophy of the media. Fourth, although it has been reported that in the aortas of SHR exist an augmented infiltration of monocyte-macrophage cells compared with the aortas of WKY,30 we did not observe such an abnormality in the aortas of adult SHR after microscopic examination. Further, the preferred substrate for oxidases in macrophages and neutrophils is NADPH rather than NADH.27
It is unclear which factors may be involved in upregulation of p22phox mRNA expression present in the aortas of adult SHR. It has been reported that hydralazine decreases both blood pressure and NADPH oxidase activity in the aortas of rats with experimentally induced hypertension.31 Thus, it is possible that the increase in blood pressure is the stimulus that induces p22phox mRNA expression in the aortas of adult SHR. However, it has been shown that norepinephrine-induced hypertension does not increase vascular NADH/NADPH oxidase activity.17 The same authors have reported that infusion of low amounts of angiotensin II that do not increase blood pressure results in the increase in this oxidase activity. Moreover, we found that p22phox mRNA expression was normal in young SHR exhibiting the same degree of elevation in blood pressure than did adult SHR. It therefore appears that long-term exposure to arterial hypertension is necessary to induce p22phox overexpression in SHR aorta. Alternatively, other nonhemodynamic factors may be also involved in this alteration (ie, tissue hormones and cytokines).
One of these candidate factors is angiotensin II. As previously mentioned, it has been found that p22phox mRNA expression was elevated in rats receiving angiotensin II infusion.31 This effect was inhibited by treatment with losartan, suggesting that it was mediated by the interaction of angiotensin II with AT1 receptors at the vascular wall. In addition, angiotensin II has been found to stimulate p22phox expression and ·O2- production in VSMCs.16 Exaggerated production of angiotensin II32 and enhanced expression of both AT1 receptor and angiotensin-converting enzyme33 have been reported in vessels of SHR compared with WKY. Furthermore, an age-dependent increase of vascular angiotensin-converting enzyme activity in SHR has been shown.34 Altogether these data would suggest that high levels of vascular angiotensin II can participate in p22phox overexpression in the aortas of adult SHR. This possibility is further supported by our findings that both p22phox mRNA expression and NADH/NADPH oxidase activity are normalized in irbesartan-treated SHR despite a noncomplete normalization of blood pressure.
It has been suggested that the development of endothelial dysfunction is linked to an exaggerated production of ·O2- in aortas from SHR.8 An enhanced production of ·O2- may result in inactivation of NO and generation of peroxynitrite.35 The resulting decrease in NO availability might be involved in the impairment of NO-dependent relaxations.2 3 8 9 10 Thus, oxidative degradation of NO caused by increased ·O2- secondary to overactivity of NADH/NADPH oxidase likely explains the diminished response to Ach observed in the aortas of adult SHR. This is further supported by the finding that responses to Ach are normal in irbesartan-treated SHR, which exhibit normal NADH/NADPH oxidase activity. Of course, our results do not exclude a role for other potential sources of ·O2- (endothelial NO synthase, xanthine oxidase) in the vascular wall of adult SHR. On the other hand, the observation that responses to SNP are altered in both untreated and treated SHR suggests that other molecular alterations (ie, diminished expression and activity of VSMC guanylate cyclase)36 37 may also contribute to impaired vasodilatory responsiveness in SHR.
On the other hand, it is possible that ·O2- is also involved in vascular hypertrophy. Recently, it has been shown that ·O2- stimulates the proliferation of VSMCs.11 In addition, a role for NADH/NADPH oxidase–derived ·O2- and H2O2 has been proposed in angiotensin II–induced VSMC growth.12 Therefore, the association of NADH/NADPH oxidase overactivity with medial hypertrophy in the aortas of adult SHR suggests a contributing role for the enzyme in vascular wall remodeling. The finding that the dimensions of the aortic wall were not completely normalized in irbesartan-treated SHR suggests that the maintenance of increased levels of blood pressure after treatment may also account for the persistence of a certain degree of aortic hypertrophy in these rats.
In summary, we reported that NADH/NADPH oxidase activity is abnormally increased in the aortic wall of adult SHR and that this abnormality is associated with upregulated p22phox mRNA expression. Our findings suggest that long-term exposure to hypertension, possibly combined with other factors (ie, local overproduction of angiotensin II), may play a role in p22phox upregulation and enzyme overactivity in the media layer from the aortas of these rats. Enhanced NADH/NADPH oxidase–mediated ·O2- production might contribute to functional and structural alterations present in the aortas of adult SHR. Further experiments are necessary to elucidate the precise mechanism through which chronic blockade of AT1 receptors prevents overstimulation of vascular NADH/NADPH oxidase in this genetic model of hypertension. The significance of these experimental results is underlined by clinical data indicating the occurrence of increased ·O2- production in humans with essential hypertension.38 39
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Acknowledgments |
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This work was supported by grant CDTI 96-0035 from the Ministry of Industry and the Ministry of Education and Culture, Spain.
Received October 11, 1999; first decision November 24, 1999; accepted December 16, 1999.
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N. Li, F.-X. Yi, J. L. Spurrier, C. A. Bobrowitz, and A.-P. Zou Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney Am J Physiol Renal Physiol, June 1, 2002; 282(6): F1111 - F1119. [Abstract] [Full Text] [PDF]
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