© 2000 American Heart Association, Inc.
UltraRapid Communications |
Regulation of Endothelial Nitric Oxide Synthase Expression by Albumin-Derived Advanced Glycosylation End Products
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
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Abstract |
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Abstract—We examined whether albumin-derived advanced glycosylation end products (AGEs) downregulate the expression of endothelial nitric oxide synthase (NOS). Significant reductions in NOS activity and cGMP levels in bovine aortic endothelial cells were observed when exposed to different concentrations of albumin-derived AGEs. Western and Northern blot analyses showed significant decreases at the protein and transcript levels. Both reductions became evident after 24 hours of exposure. Nuclear run-on assays showed that AGE-BSA did not modify the transcription rate of the NOS III gene; however, AGE-BSA treatment markedly reduced the half-life of NOS III mRNA. In addition, AGE-treated endothelial cells displayed significant reduction on their antiplatelet properties. These results indicate that NOS expression is reduced by AGEs by increasing the rate of mRNA degradation and may be relevant to the impairment of some endothelial functions observed in diabetes and aging. The full text of this article is available at http://www.circresaha.org.
Key Words: advanced glycosylation end products • nitric oxide synthase • diabetes
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelial cell–generated nitric oxide (NO) accounts for the biological activity of endothelium-derived relaxing factor. NO is derived from the guanidino nitrogen atoms(s) of the amino acid L-arginine through a reaction catalyzed by NO synthase. In endothelial cells, NO synthase (NOS III) has been characterized as a constitutively expressed calcium- and calmodulin-dependent enzyme. However, the level of NOS III expression can be altered by a growing number of different stimuli. Tumor necrosis factor-
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Cultures
Bovine aortic endothelial cells (BAECs) were prepared from aortas obtained from a slaughterhouse. Their endothelial phenotype was verified by demonstrating acetylated LDL uptake and the expression of von Willebrand factor and NOS III by immunofluorescence.
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-BSA–treated 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. Student’s 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AGE-BSA Decreases NOS III Catalytic Activity and cGMP Accumulation
We first evaluated the effects of albumin-derived AGEs on both NOS activity and cGMP levels. As depicted in Figures 1A
and 1B, we detected a marked
dose-dependent reduction in both NOS activity (from 80% to 36%) and
cGMP levels (from 66% to 33%) in BAECs exposed to increasing amounts
(0.25 to 10 µmol/L) of AGE-BSA for 48 hours. Unmodified BSA and
boiled AGE-BSA did not show any effect. There was no difference in the
effect achieved by either AGE-BSA and
NaBH4-reduced AGE-BSA, suggesting that the
effects on BAEC NOS III expression were not mediated by reversible BSA
adducts such as Amadori products.
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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-D–treated
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-BSA–treated 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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous physiological and pathophysiological stimuli are now known to increase or decrease NOS III gene transcription.20 We have found that exposure of BAECs to albumin-derived AGEs results in marked decreases in the expression of NOS III activity, protein, and transcript levels but does not affect the transcriptional rate of NOS III gene.
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 cell–derived 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.
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Acknowledgments |
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We are grateful to Dr Richard Bucala, Picower Institute for Medical Research, Manhasset, NY, for providing the rabbit antiserum (RU) to AGEs.
Received August 24, 1999; accepted January 19, 2000.
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References |
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|---|
1. Yoshizumi M, Perrella MA, Burnett JC, Lee M. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res. 1993;73:205–209.[Abstract]
2. Lu JL, Schmiege LM, Kuo L, Liao J. Downregulation of endothelial constitutive nitric oxide synthase expression by lipopolysaccharide. Biochem Biophys Res Commun. 1996;225:1–5.[Medline] [Order article via Infotrieve]
3.
Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles
IG, Moncada S. Induction of calcium-dependent nitric oxide synthases by
sex hormones. Proc Natl Acad Sci U S A. 1994;91:5212–5216.
4.
Marsden PA, Heng HH, Scherer SW, Steward RJ, Hall AV,
Shi XM, Tsui LC, Schappert KT. J Biol Chem. 1992;268:17478–17488.
5.
Arnet UA, McMillan A, Dinerman JL, Ballermann B,
Lowenstein CJ. Regulation of endothelial nitric oxide
synthase during hypoxia. J Biol Chem. 1996;271:15069–15073.
6. Phelan MW, Faller DV. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J Cell Physiol. 1996;167:469–476.[Medline] [Order article via Infotrieve]
7. Ruderman NB, Williamson JR, Brownlee M. Glucose and diabetic vascular disease. FASEB J. 1992;6:2905–2914.[Abstract]
8. Cohen RA. Dysfunction of vascular endothelium in diabetes mellitus. Circulation. 1993;87:67–76.
9. Vlassara H, Bucala R, Stricker L. Pathogenic effects of advanced glycosylation: biochemical, biologic and clinical implications for diabetes and aging. Lab Invest. 1994;70:138–151.[Medline] [Order article via Infotrieve]
10. Bucala R, Tracey KJ, Cerami A. Advanced glycosylation end products quench nitric oxide and mediate defective endothelium-dependent vasodilation in experimental diabetes. J Clin Invest. 1991;87:432–438.
11.
Vlassara H, Brownlee M, Cerami A. High affinity
receptor-mediated uptake and degradation of glucose-modified proteins:
a potential mechanisms for the removal of senescent macromolecules.
Proc Natl Acad Sci U S A. 1985;82:5588–5592.
12. Sakurai T, Takahashi H, Tsuchiya S. New fluorescence of nonenzymatically glycosylated human serum albumin. FEBS Lett. 1985;176:27–31.
13. Mitsuhashi T, Vlassara H, Founds HW, Li YM. Standardizing the immunological measurement of advanced glycation endproducts using normal human serum. J Immunol Methods. 1997;207:79–88.[Medline] [Order article via Infotrieve]
14.
Yamagishi S, Yonekura H, Yamamoto Y, Katsuno K, Sato F,
Mita I, Ooka H, Satozawa N, Kawakami T, Nomura M, Yamamoto H. Advanced
glycation end products-driven angiogenesis in vitro. Induction of
the growth and tube formation of human microvascular
endothelial cells through autocrine vascular
endothelial growth factor. J Biol Chem. 1997;272:8723–8730.
15.
Imani F, Hori Y, Suthanrhiaran M, Skolnik E, Makita Z,
Sharma V, Sehajpal P, Vlassara H. Advanced glycosylation
endproduct-specific receptors on human and rat T-lymphocytes
mediate synthesis of interferon 
: role in tissue remodeling.
J Exp Med. 1993;178:2165–2172.
16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
17.
Lamas S, Marsden PA, Li GK, Tempst P, Michel T.
Endothelial nitric oxide synthase: molecular cloning
and characterization of a distinct constitutive enzyme isoform.
Proc Natl Acad Sci U S A. 1992;89:6348–6352.
18. Greenberg ME, Bender PT. Identification of newly transcribed RNA. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl KR, eds. Current Protocols in Molecular Biology. New York, NY: John Wiley and Sons; 1996:4.10.1–4.10.11.
19.
Del Maschio A, Evangelista V, Rajtar G, Chen ZM,
Cerletti C, de Gaetano G. Platelet activation by
polymorphonuclear leukocytes exposed to chemotactic agents.
Am J Physiol. 1990;258:H870–H879.
20. Harrison DG, Sayeh H, Ohara Y, Inove N, Venema RC. Regulation of expression of the endothelial cell nitric oxide synthase. Clin Exp Pharmacol Physiol. 1996;23:251–255.[Medline] [Order article via Infotrieve]
21. Schmidt AM, Hori O, Cao R, Yan SD, Brett J, Wautier JL, Ogawa S, Kuwabara K, Matsumoto M, Stern D. RAGE: a novel cellular receptor for advanced glycation end products. Diabetes. 1996;45:S77–S80.
22. Wautier JL, Zoukourian C, Chappey O, Wautier MP, Guillausseau PJ, Cao R, Hori O, Stern D, Schmidt AM. Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats. J Clin Invest. 1996;97:238–243.[Medline] [Order article via Infotrieve]
23. Searles CD, Harrison DG, Ramasamy S. Post-transcriptional regulation of endothelial nitric oxide synthase gene expression during cell growth. Circulation. 1996;94(suppl I):I-154. Abstract.
24. Winocour PD. Platelet abnormalities in diabetes mellitus. Diabetes. 1992;41:26–30.[Abstract]
25.
Kubes P, Suzuki M, Granger DN. Nitric oxide. An
endogenous modulator of leukocyte adhesion. Proc Natl
Acad Sci U S A. 1991;88:4651–4655.
26.
Khan BV, Harrison DG, Olbrych MT, Alexander RW, Medford
RM. Nitric oxide regulates vascular cell adhesion molecule 1 gene
expression and redox-sensitive transcriptional events in human vascular
endothelial cells. Proc Natl Acad Sci
U S A. 1996;93:9114–9119.
27. Vlassara H, Fuh H, Donnelly T, Cybulsky M. Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits. Mol Med. 1995;1:447–456.[Medline] [Order article via Infotrieve]
28. 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.
29.
Ishida A, Sasaguri T, Kosaka C, Nojima H, Ogata J.
Induction of the cyclin-dependent kinase inhibitor
p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular
smooth muscle cells. J Biol Chem. 1997;272:10050–10057.
30. Hogan M, Cerami A, Bucala R. Advanced glycosylation endproducts block the antiproliferative effect of nitric oxide. Role in the vascular and renal complications of diabetes mellitus. J Clin Invest. 1992;90:1110–1115.
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