Published online before print June 21, 2001, doi: 10.1161/hh1301.092679
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Vascular Smooth Muscle Cell–Directed Overexpression of Heme Oxygenase-1 Elevates Blood Pressure Through Attenuation of Nitric Oxide–Induced Vasodilation in Mice
From the Fourth Department of Internal Medicine (T.I., T.M., S.K.), Saitama Medical School, Saitama; Department of Cardiovascular Medicine (T.S.), Graduate School of Medicine, University of Tokyo; International Medical Center of Japan (Y.Y.), Tokyo; Division of Integrative Cell Biology, Department of Embryogenesis (H.K.), Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto; and Department of Biochemistry and Integrative Medical Biology (M.S.), School of Medicine, Keio University, Tokyo, Japan.
Correspondence to Toshisuke Morita, MD, PhD, Fourth Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-Gun, Saitama 350-0495, Japan. E-mail toshijpn{at}saitama-med.ac.jp
Abstract
Abstract—To
elucidate pathophysiological roles of heme
oxygenase (HO)-1 in regulation of vascular tone in vivo, we
have developed and characterized transgenic (Tg) mice that overexpress
HO-1 site specifically in vascular smooth muscle cells (VSMCs). The Tg
mice were generated by use of human HO-1 cDNA under the control of
SM22-
promoter. The HO-1 gene overexpression was demonstrated by
Northern blot analysis and coincided with increases in the
protein expression in VSMCs and total HO activities. Tg mice exhibited
a significant increase in arterial pressure at various ages
and displayed impaired nitrovasodilatory responses in isolated aortic
segments versus nontransgenic littermates while enhancing their nitric
oxide (NO) production. The pressure of Tg mice was unchanged by
systemic administration of either
N
-nitro-L-arginine
or SNP. Furthermore, the isolated aorta in these mice exhibited lesser
extents of NO-elicited cGMP elevation via soluble guanylate
cyclase (sGC), while exhibiting no notable downregulation of sGC
expression. Such impairment of the NO-elicited cGMP increase was
restored significantly by tin protoporphyrin IX, an HO
inhibitor. On the other hand,
3-(5'-hydroxymethyl-2' furyl)-1-benzyl-indazol (YC-1), an
NO-independent activator of sGC, increased cGMP and relaxed
aortas from Tg mice to levels comparable with those from nontransgenic
mice, which indicates that contents of functionally intact sGC are
unlikely to differ between the two systems. These findings suggest that
site-specific overexpression of HO-1 in VSMCs suppresses vasodilatory
response to NO and thereby leads to an elevation of
arterial pressure.
Key Words: heme oxygenase-1 • transgenic mice • carbon monoxide • soluble guanylate cyclase • hypertension
Heme oxygenase (HO) is the rate-limiting enzyme for the degradation of protoheme IX to biliverdin, which is a precursor of bilirubin, the terminal heme-degrading product. Three isoforms of HO have been identified. HO-1 is inducible,1 whereas HO-2 and HO-3 are constitutively expressed.2 3 Carbon monoxide (CO) is another HO product of the HO reaction, which has well been recognized as a physiologically important vasoactive substance rather than a toxic waste product. This gaseous monoxide is necessary to maintain microvascular patency in the liver under both unstimulated and stress conditions.4 5 6 Inhibition of the HO/CO system with metalloporphyrins is known to lead to elevation of blood pressure and augmentation of arterial neointimal development elicited by balloon injury.7 8 On the other hand, enhancement of the HO/CO system lowers blood pressure and prevents balloon injury–induced neointimal development and hypoxia-induced pulmonary hypertension.7 9 10 Thus, acute and systemic induction of the system attenuates vascular tone and cell growth. However, it remains quite unknown whether chronic activation of the HO/CO system could cause any functional alterations in cardiovascular tone and blood pressure. Furthermore, because most pharmacological reagents that alter local HO activities such as metalloporphyrins can access tissues irrespective of cell type, mechanistic approaches to specifying cellular components responsible for the HO-1–mediated regulation of vascular tone and cell growth can hardly be addressed.
Because vascular smooth muscle cells (VSMCs) constitute a prime cellular component responsible for regulation of vascular tone and proliferative responses, we herein established transgenic (Tg) mouse strains that chronically overexpress HO-1 site-specifically in VSMCs. The current study has provided evidence that phenotypes of these mice are characterized by increased systemic blood pressure and reduced vasodilatory responses through mechanisms involving impairment of nitric oxide (NO)–mediated activation of soluble guanylate cyclase (sGC), a major receptor sensing gaseous monoxides in the cells.
Materials and Methods
All experiments were performed in accordance with the Declaration of Helsinki and were approved by the University of Tokyo and Saitama Medical School Ethical Committee for Animal Experiments.
Transgenic Construct
An SM22- promoter region that contains a 1.4-kb
fragment of the murine SM22- promoter including a 64-bp sequence of
exon 1 was isolated from mouse genomic DNA, amplified with Pfu
polymerase, and cloned into the TA cloning vector pCR TOPO
(Invitrogen). The 5' primer and 3' primer we
used for isolation are the identical sequences described previously
with an additional XhoI and
SalI as follows:
5'-AACTCGAGGGATTCAGGACGTAATCAGTGG-3' and
5'-AAGTCGACGGCTTGGTCGTTTGTGGACTGG-3',
respectively.11 An entire
protein-coding region of human HO-1 cDNA cloned into pH HO
vector12 was cut out by
digestion with HindIII and
inserted into the HindIII site
of the pBluescript sk (-) vector of which the
NotI site was converted to the
XhoI site. The
XhoI cassette of HO-1 cDNA and
a 0.85-kb SV40-derived sequence with an intron and poly-A were cloned
into the SalI and
NotI sites of the pCR TOPO
vector, respectively.
Generation and Identification of Transgenic
Mice
The 2.4-kb SM22- 5'-flanking promoter region-HO-1
cDNA construct was liberated from the vector by
XhoI digestion and purified by
agarose gel electrophoresis and a Geneclean kit (BIO101). Purified DNA
was redissolved in 5 mmol/L Tris (pH 7.4) plus 0.1 mmol/L
EDTA before pronuclear injection. Final injection concentration was
calculated as 500 copies per pL. Donor eggs were prepared from
B6C3 F1 mice. Microinjected eggs were then
transferred into the oviducts of pseudopregnant ICR foster mothers and
allowed to develop to term. Founder mice were identified by standard
Southern blot analysis of tail DNA. For Southern blot
analysis, a 300-bp fragment of human HO-1 cDNA digested with
Sph1 and
Apa1, was used as a
probe.12 Eight of the Tg
founders were fertile and were used to establish homozygous
lines.
RNA Analysis
Total tissue RNA was prepared from aorta and heart by
guanidinium isothiocyanate extraction as previously
reported.13 Fifteen
micrograms of total RNA loaded in each lane was fractionated by
formaldehyde gel electrophoresis and transferred onto nitrocellulose
membranes. The filters were hybridized with cDNA probes specific for
rat HO-1 and HO-2.14 The
mouse ß-actin gene was used for normalization of RNA
loading.14 For
quantification, we scanned autoradiographs with a laser densitometer as
described previously.14 To
detect the expression of sGC subunits 1 and
ß1 in aorta, reverse transcriptase–polymerase
chain reaction (RT-PCR) was performed using the GeneAmp RNA PCR kit,
and the primers used to amplify sGC-1 and
-ß1 and GAPDH were as described
elsewhere.15 The amplified
cDNAs were size-fractionated and stained with ethidium bromide. The
fluorescence was determined
densitometrically.
Determination of Heme Oxygenase
Activity
Heme oxygenase activity in microsomes was
determined in aorta, femoral artery, and heart (left ventricle) from Tg
mice as compared with that in age-matched nontransgenic (Ntg)
littermates.16 The activity
was expressed as nmol bilirubin formed per hour per mg of protein. The
protein concentration was determined by a dye binding assay
(Bio-Rad).
Immunohistochemistry
HO-1 immunoreactivity was detected in aorta using
rabbit polyclonal antibody against human HO-1 (1:1000 dilution)
purchased from StressGen. Briefly, sections were trypsinized with 1%
vol/vol trypsin in PBS at 37°C for 60 minutes and washed with PBS.
Nonspecific protein binding was blocked with 0.1% vol/vol horse normal
serum at room temperature for 30 minutes. The sections were then
incubated overnight at 4°C with the primary antibody to visualize
immunoreactivities.7
Western Blot Analysis
Frozen tissue was homogenized in lysis
buffer (containing, in mmol/L, NaCl 150, Tris [pH 7.5] 30, and
PMSF 1; 0.25 mol/L sucrose; 5 µg/mL leupeptin; and 1.9 µg/mL
aprotinin). Protein concentrations were determined with a dye binding
assay (Bio-Rad) using BSA as a standard. Total
tissue protein (50 µg) was prepared in sample buffer for Western blot
analysis.9 Polyclonal
antibodies against endothelial NO synthase (eNOS) and
sGC were purchased from StressGen and
Calbiochem, respectively.
Measurements of Blood Pressure
Male Tg mice (6 to 14 weeks old) and their Ntg
littermates used in the current study were obtained from heterozygote
intercrosses. Systolic blood pressure (SBP) and heart rate were
measured both directly and indirectly. For indirect measurements, SBP
and heart rate were measured without anesthesia using a
programmable sphygmomanometer connected with cuff probe for mouse
(MCP-1, Softron) as described
previously.17 For direct
measurements, we anesthetized mice with an
intraperitoneal injection of pentobarbital sodium
at 0.08 mg/g body weight and cannulated their right femoral artery with
polyethylene tubing. After mice were allowed to recover, blood pressure
was recorded continuously with the mice conscious and unrestrained
as described elsewhere.18
When necessary, sodium nitroprusside (SNP) was administered through the
femoral vein catheter.
Determination of
NO2- and
NO3- (NOx)
Concentrations in Urine
Mice were placed in individual metabolic
cages for 24 hours for collecting urine, and concentrations of NOx were
measured with a commercial kit (Cayman) before and after treatment with
N-nitro-L-arginine
(L-NNA) for 2
weeks.7
Ex Vivo Studies of Aortic Tone
The descending aorta was carefully removed and a
cylindrical segment (3 mm long) was excised from aorta. The rings
were equilibrated in an organ bath for 30 minutes under a resting
tension of 0.3 g in carbogenated (95%
O2/5% CO2) Krebs
bicarbonate solution, pH 7.4 (containing, in mmol/L, NaCl 120, KCl
5.2, CaCl2 2.4,
MgSO47H2O 1.2,
NaHCO3 25, Na2-EDTA 0.03,
and dextrose [pH 7.4] 11). Subsequently, rings were contracted with
phenylephrine (1 µmol/L), and the relaxant responses to a
cumulative dose of acetylcholine (Ach); SNP; or
3-(5'-hydroxymethyl-2' furyl)-1-benzyl-indazol (YC-1), an
allosteric reagent for sGC that activates the enzyme through
NO-independent mechanisms,19
were assessed. The relaxation responses were expressed as mean±SEM of
percentage values showing reversal of the
phenylephrine-induced contraction
responses.
Determination of cGMP Content
Concentrations of cGMP were measured in extracts of
aorta using a commercial ELISA kit (Amersham).
Aortic rings were incubated for 15 minutes with vehicle,
S-nitrosoglutathione (GSNO; 50
µmol/L); SNP (300 nmol/L and 10 µmol/L); tin protoporphyrin IX
(SnPP; 10 µmol/L and 1 mmol/L), an HO inhibitor; or
YC-1 (30 µmol/L). At the end of experiments, samples were immediately
frozen in liquid nitrogen and stored at -80°C until the
cGMP assay was
performed.15
Assessment of sGC Activity in Aortic
Protein Extracts
After preparation of the aorta, samples were
incubated in Krebs buffer (37°C in 5% CO2) in
the presence or absence of the indicated reagents. After 15 minutes,
the aorta was shock frozen, homogenized in liquid nitrogen,
and dissolved in 200 mL of ice-cold lysis buffer (containing, in
mmol/L, Tris-HCl 20, sucrose 0.25, EDTA 200, DTT 10, and benzamidine 2,
and 10 µg/mL leupeptin). The sample was cleared through
centrifugation
(13 000g for 30 minutes) to
determine the GC
activities.20
Reagents
All reagents were purchased from
Sigma, unless otherwise specified. L-NNA and
amlodipine were dissolved in drinking water to give a final dose of 60
and 5 mg/kg per day for 2 weeks, respectively. Clotrimazole (80
mg/kg body weight, IP), a reagent that blocks cytochrome P450
monooxygenases, was suspended in corn oil for
administration once every 24
hours.21 SNP was dissolved
with saline and administered intravenously at desired
concentrations.22 When
necessary, aortic rings were incubated with SnPP (Porphyrin
Products) or L-NNA as described
previously.14
Data Analysis
Results are expressed as mean±SEM. Unless otherwise
stated, each of the studies was performed in a minimum of 10 to 14
mice. Statistical significance was assessed by one-way ANOVA combined
with the Fisher multiple comparison test.
P<0.05 was considered
statistically significant.
Results
Establishment of the HO-1 Tg Mice
The Tg mice in the current study overexpressed
HO-1 under the control of SM22- promoter. The construct was shown in
Figure 1A
, and the restriction fragment comprising
promoter and HO-1 cDNA was purified and used for
microinjection. Microinjection of the transgenic construct into
fertilized mouse eggs gave rise to 28 live-born offspring. Among them,
8 were founders carrying the transgene; the founders were identified by
Southern blot analysis of genomic DNA. The copy number of the
transgene incorporated into the germline varied from one to nine as
estimated by slot hybridization. Three independent lines with the
highest copy numbers (
7 to 9) were selected for further
analysis and characterization as described below.
Figure 1B showed Northern blot analysis of HO mRNA
in aorta and heart from Ntg and Tg mice. The HO-1 gene in aorta from Tg
mice expressed 5- to 6-fold greater than that from Ntg without any
changes in HO-2 expression, whereas there were no notable changes in
message levels in the heart between Ntg and Tg mice. This correlated
with a 4- to 5-fold induction of the HO-1 protein as judged by Western
blot analysis in aorta from Tg mice
(Figure 1C) and coincided with an increase in the HO activity
in vessels from Tg as compared with Ntg
(Figure 1D). Immunohistochemistry revealed that the HO-1
immunoreactivities were evident in the medial smooth muscle layer of
aorta from Tg mice, whereas there were few if any in the Ntg aorta from
Ntg
(Figure 1E).
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HO-1 Tg Mice Exhibit High Blood
Pressure
The Tg mice showed no changes in appearance and no
changes in body weight at 6 weeks of age (20.0±1.2 g [Tg] versus
20.1±1.1 g [Ntg]). SBP of 6-, 10- and 14-week-old mouse was measured
both indirectly and directly. SBP of Tg mice as measured by tail cuff
was significantly higher than that of Ntg littermates, and this result
was reproducible when SBP was measured directly at all indicated time
periods (indirect and direct measurements, respectively, were 118±6
and 116±6 mm Hg [Tg] versus 94±4 and 92±3 mm Hg
[Ntg] at 6 weeks; 120±4 and 118±3 mm Hg [Tg] versus 94±3
and 92±4 mm Hg [Ntg] at 10 weeks; and 118±4 and 116±3
mm Hg [Tg] versus 94±5 and 92±4 mm Hg [Ntg] at 14 weeks;
P<0.01). However, no
significant difference in heart rate was observed at all indicated
periods (data not shown). We therefore analyzed the
parameter using 6-week-old mice for further
analyses of contractile function of aorta.
Responses of Blood Pressure to L-NNA and SNP
Are Reduced in Tg Mouse
Two-week treatment with amlodipine normalized blood
pressure in Tg mice to the levels of Ntg mice (before, 116±4
mm Hg; after, 94±3 mm Hg). Treatment with clotrimazole
caused a slight decrease in blood pressure of Ntg and Tg mice, but with
no statistical significance as compared with the Ntg group
(Figure 2A). L-NNA administration increased blood
pressure specifically in Ntg but not in Tg mice. As a result of the 1-
and 2-week treatment, difference in blood pressure between the two
groups was canceled out
(Figure 2A). Intravenous administration of SNP
(24 to 192 nmol/kg) reduced SBP in Ntg mice, but the response of the
decreased pressure was significantly blunted in Tg mice. As seen in
Figure 2B, the reduction of SBP in Ntg mice peaked as early
as 30 seconds after injection and lasted within 5 minutes at all doses,
whereas that in Tg mice was markedly suppressed and did not last more
than 3 minutes. These results suggest that nitrovasodilatory responses
were significantly reduced by the VSMC-directed overexpression of
HO-1.
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Increased NO Generation and Overexpression of
eNOS in Aorta and Kidney of Tg Mice
To examine whether reduced production of NO
could result in the increased blood pressure of Tg mice, we measured
urinary excretions of NOx before and after the 2-week treatment with
L-NNA. As seen in
Figure 3A, the basal urinary excretion of NOx from Tg mice
was significantly greater than that of Ntg mice. This event coincided
with the increased expression of eNOS protein in aorta and kidney as
judged by Western blot analysis
(Figure 3B). At 14 days after L-NNA treatment, urinary NOx
levels were not significantly different between both strains
(Figure 3A).
|
Desensitization of NO-Mediated Vasorelaxation
in Tg Mice
To examine mechanisms for impairment of
nitrovasodilatory response of Tg mice,
endothelium-dependent and
endothelium-independent relaxation of aortic rings
precontracted with phenylephrine were tested.
EC50, which indicates the negative logarithm of
50% relaxation of the aortic ring segment for Ach and SNP, was
significantly greater in Tg as compared with Ntg mice (6.21±0.03
[Ach] and 6.84±0.03 [SNP] in Tg mice versus 5.9 5±0.02 [Ach]and
5.99±0.2 [SNP] in Ntg mice; n=10;
P<0.01). In contrast, the
EC50 for YC-1 was not significantly different
between Tg and Ntg mice (6.18±0.05 in Tg and 6.02±0.04 in Ntg mice;
n=10; P>0.05)
(Figure 4). These results suggest that the impaired
vasodilator response of Tg results from reduced sensitivity of sGC to
NO rather than from decreased bioavailability of sGC by
itself.
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Reduction of cGMP Contents in Aorta of Tg
Mice
We found that basal contents of cGMP in aorta from Tg
mice were significantly reduced as compared with those measured in Ntg.
Treatment with L-NNA decreased cGMP contents specifically in Ntg mice,
but not in Tg mice. As a result, cGMP contents in Tg mice were
significantly greater than those in Ntg mice. The treatment with SnPP
dose-dependently increased aortic cGMP contents in Tg to levels similar
to those observed in control Ntg mice. Treatment with GSNO or SNP did
not cause a notable increase in aortic cGMP contents in either control
Tg or amlodipine-treated Tg mice as compared with that observed in Ntg
mice
(Figure 5B). However, blockade of the HO reaction by
SnPP significantly and dose-dependently restored the SNP-induced cGMP
elevation in the Tg aorta, although L-NNA did not improve it. On the
other hand, treatment with YC-1 resulted in a marked increase in cGMP
levels in Tg, which was comparable with that in Ntg
(Figure 5B). These results suggest that contents of
functionally intact sGC were unchanged in Tg, whereas the sensitivity
of the enzyme to NO was impaired through mechanisms involving the
catalytic activities of HO.
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SnPP Restored the Reduced sGC Activity in Aorta
From Tg Mice
The basal and SNP-stimulated sGC activities in aorta
from Tg mice were significantly reduced as compared with Ntg mice
(Figure 6A), although amounts of sGC expressed in situ were
almost identical between Tg and Ntg mice, as judged by RT-PCR and
Western blot analysis
(Figure 6B). SnPP dose-dependently restored the
SNP-induced increase in the sGC activity in aorta from Tg mice.
Incubation with catalase (500 U/mL) or superoxide dismutase (100 U/mL)
did not improve SNP-induced increase in the sGC activity
(Figure 6A).
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Discussion
In this study, we have established Tg mice that
overexpress HO-1 specifically in VSMCs. To overexpress the exogenous
human HO-1 gene in a VSMC-selective manner, we have used the mouse
1.4-kb SM22- promoter region. SM22- is known to be a
VSMC-specific protein, and its expression in adult is restricted to
smooth muscle–containing tissues, whereas in the early stages of
development, it is also present in skeletal and cardiac muscle
tissues.23 Experiments using
transgenic mice harboring SM22-–lacZ transgenes revealed that
transgene expression was changed in a time course similar to
those of endogenous
SM22-.11 The Tg mice
herein established turned out to express markedly HO-1 gene and protein
in vessels with 3-fold increase in its catalytic activity.
Immunohistochemistry revealed that intensive HO-1 immunoreactivities
occur in VSMCs, but not in endothelial cells,
indicating that SM22- promoter used in the current study worked
successfully. These findings indicate that Tg mice overexpress HO-1 in
a VSMC-specific manner concurrently with an actual increase in the
catalytic activity.
Observation that the site-specific HO-1 gene transfer into VSMCs reduces nitrovasodilatory responses led us to shed light on a novel biological effect of CO on the NO-mediated vasorelaxing mechanisms. Vasoactive properties of CO derived from HO have been shown to be ascribable to its ability to relax smooth muscle cells by activating sGC; this enzyme contains a prosthetic heme that acts as the receptor not only for NO but also for CO. However, previous investigation in vitro showed that NO and CO activate the cyclase by distinct mechanisms.24 NO binds to the heme and proceeds to break the proximal histidine-iron bond, forming a 5-coordinate nitrosyl heme complex that is thought to result in conformational changes and a 100-fold increase in cGMP generation with its excess amounts. By contrast, CO, while binding to the heme of the cyclase with high affinity, forms a 6-coordinated heme complex with the histidine-iron bond remaining intact.24 The potency of CO is thus far less than that of NO. Considering such a discrepancy of the cyclase-activating properties between the gases, the current results led us to hypothesize that CO endogenously generated from VSMCs could modulate the cyclase activities through competing with NO released from endothelial cells. Such a hypothesis is supported by the observation that vasorelaxation mediated by NO was significantly suppressed, whereas the expression of the cyclase seems unchanged in the HO-1 Tg mice. Another possible mechanism by which HO-1 overexpressed in VSMCs counteracts NO-mediated vasorelaxation is involvement of accelerated degradation of heme required for the cyclase in VSMCs. However, this scenario appears implausible, because the VSMCs were able to upregulate sGC activities normally in response to YC-1, the reagent sensitizing the enzyme to endogenous CO.25 In other words, sGC expressed in the arterial system of Tg mice appears to be functionally intact, and the CO-mediated impairment of vasorelaxation is an NO-dependent event. CO overproduced through the HO reaction has recently been shown to alter cytochrome P450–mediated synthesis of vasoactive prostanoids6 and could also explain the alterations in systemic blood pressure. This possibility is also unlikely, inasmuch as the treatment with the enzyme inhibitor clotrimazole exhibited no effects, at least in the current study. These facts led us to hypothesize that CO competes with NO on sGC and may function as a partial agonist for the enzyme; this gaseous monoxide stimulates the enzyme when NO levels are low (eg, in liver microcirculation),4 16 whereas it inhibits the enzyme when NO levels are sufficient. Such a possibility was previously discussed elsewhere26 and is supported by the present results showing differences in aortic cGMP contents between Tg and Ntg mice; mechanisms for a decrease in aortic cGMP contents in Tg mice could be ascribable to a partial blockade of NO through CO overproduction. Tg mice did not greatly alter the cGMP contents on administration of the NO synthase (NOS) inhibitor, because endogenously overproduced CO restores the cyclase activity. On the other hand, the treatment of the same inhibitor reduced the cGMP contents markedly in Ntg mice because of a relative shortage of CO. Furthermore, in vitro blockade of HO by the enzyme inhibitor restores the cGMP contents in Tg mice, because CO-mediated blockade of NOS is canceled.
Although previous studies using disease models have not fully addressed cellular components responsible for the HO-1 induction, microvascular endothelial cells have been shown to induce HO-1 on inflammatory stimuli and could alter adhesivity of inflammatory cells.27 On the other hand, atherosclerosis has been shown to account for such a disease condition that causes the HO-1 induction in cells of the arteriolar wall.28 It has also been shown that VSMCs constitute a major site for the HO-1 induction in the model of arterial balloon injury.7 These findings indicate that the cellular site responsible for the enzyme induction and subsequent biological effects on vascular functions could vary among different disease models. Because of such a multiplicity and heterogeneity of the HO-1 induction, it has been difficult to address precisely the consequences of HO-1 overexpression on functions of vascular systems in vivo. Thus, previous methods to induce or overexpress HO-1 through pharmacological interventions or nonspecific gene transfer into tissues have not allowed us to examine consequential outcomes of the enzyme induction.
In this respect, the current genetic engineering approach provided evidence for HO-1–mediated functional remodeling of NO-mediated regulation of the arteriolar tone that is triggered primarily through VSMCs. It should be noted that eNOS is upregulated concurrently with elevated urinary excretions in Tg mice, although mechanisms for this event are unknown. The induction of HO-1 has been known to alter local NO generation through multiple mechanisms, as follows: competitive consumption of NADPH between the two enzyme systems, degradation of the prosthetic heme for NOS, and CO binding to the heme.5 However, all of these mechanisms did not explain upregulation of endothelium-derived NO generation in the Tg mice. Considering that local wall shear stress plays a crucial role in the expression of eNOS,29 it is not unreasonable to speculate that the elevated NO generation in Tg mice could result from reduced sensitivity of sGC to endothelial NO and subsequent impairment of vasodilation that causes in turn the elevated shear force in the arterial endothelium. Such interactions between endothelial NO generation and HO-1 in VSMCs deserve further study given the evidence for differences in local hemodynamic forces between Tg and Ntg mice.
Finally, the current study shed light on clinical implications for HO-1–mediated desensitization of sGC to NO in the vascular systems that may serve as a putative mechanism for resistance to nitrovasodilators. Previous studies have shown that exposure to greater doses of exogenous NO decreases the sensitivity of VSMCs to NO donors through downregulating amounts of the cyclase expression.30 31 However, considering that excess amounts of NO could induce HO-1 in various experimental models,5 the current study raised a possibility that such an NO-mediated desensitization of the cyclase could occur through CO-mediated mechanisms even when the expression amounts of sGC are not downregulated. Further investigation as to whether repeated administration of nitrovasodilators could induce HO-1 in and around VSMCs should obviously be necessary to address roles of the HO/CO system in the modulation of NO-mediated vascular responses in human cases. Our mouse model provided new insights into the in vivo mechanisms of cross talk between NO and CO for regulation of the activity of sGC, serving as a useful model to explore the pathophysiological roles of CO in cardiovascular diseases.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education of Japan, a grant from Maruki Memorial Foundation, and by the Japan Cardiovascular Disease Research Foundation (to T.M.). We thank Junko Tatebe for her technical assistance.
Footnotes
Original received December 11, 2000; revision received May 9, 2001; accepted May 9, 2001.
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