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From the Laboratorio de Farmacología (A.R., D.G., B.H., R.D., K.O.), Centro de Química Farmacéutica, Apartado Postal 6990, La Habana, Cuba; Laboratorio de Bioquímica (S.R.), Centro Nacional de Investigaciones Científicas, Apartado Postal 6880, La Habana, Cuba.
Correspondence to Armando Rojas, Centro de Química Farmacéutica, Ave 21 esquina a 200, Apartado Postal 6990, La Habana, Cuba. E-mail ily{at}biocnic.cneuro.edu.cu
| Abstract |
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Key Words: advanced glycosylation end products nitric oxide synthase diabetes
| Introduction |
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1 and
lipopolysaccharide2 decrease expression of NOS
III. In contrast, estrogens3 and shear
stress4 can increase NOS III expression. The effect of
hypoxia is less clear; some authors have reported either an
increase5 or decrease6 in NOS III mRNA. Vascular diseases account for the majority of the clinical complications of diabetes mellitus.7 Although the molecular basis of the mechanisms involved for the development of vascular disease in diabetes mellitus is poorly understood, several lines of evidences demonstrate impairment in endothelium-dependent relaxation of diabetic blood vessels.8
Reducing sugar such as glucose can react nonenzymatically with the amino groups of proteins to form complex structures called advanced glycosylation end products (AGEs). In normal aging processes and in some pathological conditions such as diabetes, the excessive accumulation of AGEs could lead to tissue dysfunction.9 Recently, AGEs have been shown to quench NO and, therefore, may play a role in the defective endothelium-dependent vasodilatation in experimental diabetes.10
To our knowledge, the effect of AGEs on NOS III expression is unknown. We have undertaken the present study to test the hypothesis that AGEs reduce the expression level of NOS III. We show a significant decrease in the amount of NOS III protein and mRNA in AGE-exposed bovine endothelial cells.
| Materials and Methods |
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Preparation of AGE-Modified Albumin
AGE-BSA was prepared as described.11 Such treatment
has been shown to be sufficient for the accumulation of large amounts
of AGE on albumin.11 12 We verified the presence
of AGE structures in our preparation of AGE-BSA by a direct ELISA using
a specific antiserum to a common AGE immunological epitope found both
in vitro and in vivo as described.13 We ruled out the
effect of early glycosylation products by incubating AGE-BSA with
200-fold molar excess of sodium borohydride.14 15
NOS Catalytic Activity
BAECs exposed to different concentrations of AGE-BSA for 48
hours were harvested, sonicated, and centrifuged at
1000g for 5 minutes. NOS activity in total cell lysates (100
to 150 µg of total proteins) was measured by the conversion of
[14C]arginine to
[14C]citrulline.
Determination of cGMP
BAECs (106 cells) were challenged with
0.5 mmol/L isobutyl-1-methylxanthine at the time of AGE-BSA
addition. The content of cGMP was measured using a specific binding kit
according to the recommendations of the supplier (Amersham).
Western Blot Analysis
Total protein (20 µg) was electrophoresed on 7.5%
SDS-polyacrylamide gels and transferred to a nylon membrane.
The membrane was blocked with 5% skim milk, incubated with a
monoclonal antibody against NOS III (Transduction Labs), washed,
incubated with sheep anti-mouse Ig conjugated with horseradish
peroxidase, and developed with a chemiluminescent reporter system (ECL,
Amersham).
Northern Blot Analysis
Total RNA was isolated by the guanidinium
thiocyanate-phenol-chloroform method.16 Fifteen micrograms
was separated by electrophoresis on a 1% agarose-MOPS gel with 2.2
mol/L formaldehyde and transferred to a nylon membrane by capillary
action. Membranes were hybridized with a cDNA probe specific for bovine
NOS III17 labeled with [
-32P]
dCTP. After overnight hybridization, blots were washed and exposed to
an x-ray film (Hyperfilm, Amersham) for 48 hours. Human GAPDH was used
to probe the GAPDH message. Quantification of
autoradiographic results was performed with a laser
densitometer and ImageMaster software.
In Vitro Elongation of Nascent RNA (Run-on Assay)
Nuclei from 108 BAECs were prepared and in
vitro transcription with 32P UTP was performed
essentially as described.18 cDNAs for NOS III and
ß-tubulin were used as probes. pBlueScript I plasmid DNA was used as
a control.
Platelet Aggregation
Human-washed platelets were prepared as
described.19 Briefly, 108
platelets were incubated in the presence of control and AGE-treated
BAECs. Under these experimental conditions, 106
control BAECs was the proportion able to inhibit the 50% of
platelet aggregation induced by 40 mU/mL thrombin. Control and
AGE-BSAtreated BAECs were added 2 minutes before the addition of
thrombin.
Statistical Analysis
All values are presented as mean±SEM of multiple
experiments. Comparisons between multiple treatment groups of changes
in NOS activity and cGMP levels were made by ANOVA and Newman-Keuls
test. Students t test (two-tailed) for unpaired data was
used to determine statistical significance in the platelet
aggregation studies. Differences were considered significant at
P<0.05.
| Results |
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AGE-BSA Decreases NOS III Expression
We next examined whether NOS-specific activity reduction was due
to a decrease in NOS III expression. AGE-BSA (0.25 to 10 µmol/L)
caused a concentration-dependent decrease of NOS III protein in BAECs
exposed for 48 hours (Figure 2A
).
Furthermore, NOS III protein levels were unchanged during the first
12-hour period. At 24 hours, NOS III protein reduction became evident
and was markedly decreased after 48 hours (Figure 2B
). Exposure
of BAECs to AGE-BSA for longer periods (72 to 96 hours) did not result
in further decreases in NOS III protein levels (data not shown).
Additionally, NOS III protein decrease was paralleled by a
reduction of NOS III mRNA (Figure 3
). It
is noteworthy that exposures for periods shorter than 24 hours did not
modify the NOS III transcript levels.
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AGE-BSA Has No Effect on the Transcriptional Rate of the NOS
III Gene
To explore the mechanism by which AGE-BSA decreases the
steady-state NOS III mRNA levels, nuclear run-on experiments were
conducted to ascertain whether AGE-BSA attenuates the transcription of
NOS III gene. As shown in Figure 4
, AGE-BSA did not affect NOS III gene transcription.
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AGE-BSA Changes NOS III mRNA Stability
In view of the preceding results, we next examined the effects of
AGE-BSA on the half-life of NOS III transcript. Actinomycin-Dtreated
BAECs were cultured for 24 hours in the presence or absence of 10
µmol/L of AGE-BSA, before harvesting of total RNA. As can be seen in
Figure 5
, actinomycin-D did not modify
the hybridization signals of NOS III under control conditions (no
AGE-BSA treatment) over 24 hours. However, the presence AGE-BSA
drastically shortened the half-life to less than 20 hours.
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AGE-BSA Decreases Antiplatelet BAEC Function
To further investigate the physiological
consequences of NOS III downregulation by AGE-BSA, we evaluated the
effects on the antiplatelet properties of BAECs, on the basis of
BAEC-derived NO production. As expected, AGE-BSA treatment
markedly modified BAEC antiplatelet properties. In our experimental
model, 106 BAECs were needed to reduce
thrombin-induced platelet aggregation by 50%, whereas the same
amount of 10 µmol/L AGE-BSAtreated cells reduced platelet
aggregation by only 18%, as depicted in Figure 6
. Treatment with
heat-inactivated AGE-BSA did not modify the
antiplatelet properties of BAECs.
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| Discussion |
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The major mechanism involved in downregulation of NOS III expression
seems to be due to an enhanced rate in mRNA degradation, as
demonstrated for both tumor necrosis factor-
and
lipopolysaccharide.1 2 In the present study,
we show that an increase in mRNA degradation rather than a decrease in
transcription mediates the inhibition of NOS III gene expression.
The decrease of NOS III mRNA by AGE-BSA requires at least 24 hours to
become apparent. This long lag time suggests that the reduction of NOS
III transcripts by AGE-BSA may involve another factor. Although the
identity of newly transcribed gene product(s) required for the
observed effects remains to be established, it is noteworthy that
oxidant stress and, consequently, the activation of nuclear factor-
B
are observed once AGE binds to its receptor.21 Both events
seem to be involved in early vascular hyperpermeability observed in
diabetic rats.22 More interestingly, two proteins have
been identified to bind to the 3' untranslated region of NOS III mRNA
destabilizing it.23 Destabilization of NOS III mRNA by
AGE-BSA could result from a protein-mRNA interaction, which would allow
the degradation elements in the 3' untranslated region to become
active. This would be an interesting approach to investigate further
into the molecular mechanism(s) of NOS III mRNA destabilization induced
by AGE-BSA.
It is noteworthy that platelet hyperactivity is a typical feature of the diabetes-associated prothrombotic state.24 The fact that AGE-BSA markedly reduced the antiplatelet activity of endothelial cells, mainly because of the reduction of NOS III expression, may be an additional factor to aggravate vascular complications. Inhibition of endothelial cellderived NO enhances polymorphonuclear leukocyte adherence and emigration in postcapillary venules as demonstrated by Kubes et al,25 suggesting the role of NO in preventing leukocyte-endothelial cell adhesion. Furthermore, NO decreases cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1).26 Recently, aortic tissue AGE accumulation has been associated with a significant increase in expression of VCAM-1 and ICAM-1.27
In addition, vascular smooth muscle cell proliferation is clearly associated with the accelerated vasculopathy observed in diabetes. It has been demonstrated that NO mediates the cytostasis of subendothelial vascular smooth muscle cells,28 29 and AGEs may favor cell proliferation by quenching endothelium-derived NO30 and thus plays a significant role in the onset of proliferative vascular lesions.
In summary, AGE-BSA markedly reduces NOS III expression, which may represent a new insight into the vascular complication observed in diabetes. Whether downregulation of NOS III expression has any effect on impaired endothelium-dependent relaxation must await further investigation. Thus, a better understanding of such a mechanism may lead to new strategies in the prevention of vascular dysfunction.
| Acknowledgments |
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Received August 24, 1999; accepted January 19, 2000.
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