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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2003 as doi:10.1096/fj.02-0490fje. |
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* Center for Cardiovascular Biology and Medicine, King’s College London, London SE1 1UL, UK; and
* Diabetic Microangiopathy Research Unit, Villeurbanne, France
2Correspondence: GKT School of Medicine (Cardiovascular Division), Room 2.36B New Hunts House, King’s College London (Guy’s Hospital Campus), London Bridge, London SE1 1UL, UK. E-mail: albert.ferro{at}kcl.ac.uk
SPECIFIC AIMS
Advanced glycation end products (AGE), formed by the nonenzymatic reaction of glucose and other reducing sugars with amino groups on proteins and other macromolecules, are generated in increased amounts during chronic hyperglycemia and are believed to contribute significantly to the vascular disease seen in diabetes mellitus. In the present study, we investigated whether albumin AGE-modified by glucose (AGE-Glu) can give rise to vascular endothelial dysfunction by examining its effects on the nitric oxide (NO) system both in vivo after injection into rabbit femoral artery and in vitro on isolated rabbit thoracic aortic rings and on cultured human umbilical vein endothelial cells (HUVEC).
PRINCIPAL FINDINGS
1. N-Carboxymethyllysine (CML, the major AGE adduct found in vivo) was determined in the plasma of diabetic patients (types 1 and 2, n=20) and of healthy controls of similar age and sex (n=13) by competitive CML-specific ELISA
Diabetics diagnosed 6.7 ± 1.7 years previously had a glycated hemoglobin (HbA1C) of 7.9 ± 0.9% and significantly higher plasma glucose (random) than controls (11.6±1.4 vs. 5.5±0.9 mM, P<0.001). Concentrations of CML were 32.6 ± 8.3 units/mL in plasma of the diabetic patients compared with 3.5 ± 2.1 units/mL in that of the controls (P<0.01).
2. AGE-Glu (20 mg/mL) was prepared by incubating bovine serum albumin with glucose for 12 wk; CML was quantified by competitive CML-specific ELISA
AGE-Glu contained 150 CML units/mg protein whereas unmodified albumin contained 1.95 CML units/mg protein. We therefore examined the effects of AGE-Glu 50, 100, and 200 µg/mL (corresponding to 7.5, 15, and 30 CML units/mL) on vascular endothelial function, since such concentrations are representative of those found in the circulation of diabetic patients.
3. Treatment of rabbit aortic rings with AGE-Glu at concentrations ranging from 50 to 200 µg/mL inhibited relaxant responses to acetylcholine (ACh, an endothelial NO-dependent vasodilator), but not those to sodium nitroprusside (NP, an endothelium-independent vasodilator that liberates NO by spontaneous degradation), after norepinephrine preconstriction
The effect of AGE-Glu on ACh responses was concentration dependent, such that at the highest concentration of AGE-Glu used (200 µg/mL), Emax to ACh, was decreased to 
50% of control. Unmodified albumin at these same concentrations had no effect on ACh-mediated vasorelaxation.
The inhibitory effect of AGE-Glu on ACh-mediated vasorelaxation was found to be reversible, since washout of the AGE-Glu resulted in a restoration of the ACh relaxant responses to control values. MnTMPyP 100 µg/L, a cell-permeable superoxide dismutase mimetic and oxygen-derived free radical scavenger, did not prevent the inhibition of ACh-induced vasorelaxation by AGE-Glu. Moreover, coincubation of vascular rings with a rabbit polyclonal antibody specific for the CML epitope prevented the impairment of ACh-mediated vasorelaxation by AGE-Glu, whereas nonimmune rabbit serum had no such effect.
4. In rabbit femoral artery in vivo, an abrupt 40% increase in perfusion rate induced a sudden increase in perfusion pressure, followed by a gradual decline back to baseline over the ensuing 20 min that was inhibited by local administration of NG-nitro-L-arginine methyl ester (L-NAME, a NO synthase [NOS] inhibitor) 1 µmol/kg, consistent with acute shear stress-induced NO release
We found that AGE-Glu 200 mg/kg prevented the decline of pressure toward baseline after the initial increase as effectively as L-NAME, whereas unmodified albumin 200 mg/kg had no such effect, again suggesting an inhibition of vascular NO production by AGE-Glu.
5. To ascertain whether the effect of AGE-Glu on endothelium-dependent relaxation could be explained by inhibition of NO synthesis, we examined the effect of AGE-Glu on NOS activity in cultured HUVEC, as determined by conversion of L-[3H]arginine to L-[3H]citrulline
These cells were exposed to AGE-Glu (200 µg/mL) or vehicle for 30 min; L-[3H]arginine to L-[3H]citrulline conversion was then determined in the absence or presence of 10-5M histamine, a known activator of NOS in endothelial cells. AGE-Glu had no significant effect on basal L-[3H]citrulline production. Histamine increased L-[3H]citrulline production by 33.4% (P<0.001), and this increase was abolished by AGE-Glu at a concentration of 200 µg/mL (Fig. 1
). Unmodified albumin at the same concentration, by contrast, had no effect on the histamine-induced increase in L-[3H]citrulline formation. Anti-CML antibody at a dilution of 1:1000 prevented the inhibitory effect of AGE-Glu on histamine-induced L-[3H]citrulline production by HUVEC (Fig. 1)
. Similarly, antibody to the AGE-R1 receptor, previously shown to be expressed by vascular endothelial cells, abolished the inhibitory effect of AGE-Glu on histamine-induced L-[3H]citrulline production, again at a dilution of 1:1000 (Fig. 1)
. Nonimmune rabbit serum at the same dilution, by contrast, had no such effect.
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6. Effects on L-[3H]citrulline production observed in HUVEC were not explained by AGE-Glu cytotoxicity, as demonstrated by Trypan blue dye exclusion
Exposure of HUVEC to AGE-Glu (100 or 200 µg/mL) did not affect cell viability for up to 24 h. At 72 h, however, cell viability decreased relative to control cells, an effect that was prevented by coincubation with either antibody to CML or antibody to AGE-R1 (each at a dilution 1:1000) but not by normal rabbit serum.
7. Western blot studies of the endothelial isoform of NOS (eNOS) isolated from lysed HUVEC by immunoprecipitation after exposure of the intact cells to AGE-Glu (200 µg/mL) or vehicle for 30 min in the absence or presence of antibody to CML or antibody to AGE-R1 (each at a dilution 1:1000) demonstrated no change in the amount of eNOS expressed after AGE-Glu treatment
On the other hand, a decrease was observed in phosphoserine content of eNOS in HUVEC after treatment with AGE-Glu (200 µg/mL), which was prevented by coincubation of cells with antibody to either CML or AGE-R1 (Fig. 2
). Enzymic activity of immunoprecipitated eNOS, as determined by L-[3H]arginine to L-[3H]citrulline conversion in the absence and presence of L-NAME 0.1 mM, was abolished by incubation of HUVEC with AGE-Glu (200 µg/mL) compared with control incubations; this effect was again prevented by coincubation of cells with antibody to either CML or AGE-R1 (dilution 1:1000 for each).
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CONCLUSIONS AND SIGNIFICANCE
Our results demonstrate that AGE-Glu, at concentrations reflecting those of CML found in the plasma of diabetic patients, exerts profound effects on vascular function, secondary to inhibition of endothelial cell NO generation. It inhibits vasorelaxation to ACh in rabbit aorta, with no effect on vasorelaxation to NP. It abolishes the NO-dependent vasorelaxant response to shear stress in rabbit femoral artery in vivo. CML residues are implicated, since anti-CML antibody abolishes the effect of AGE-Glu on ACh responses in rabbit aorta. The impairment of ACh-mediated vasorelaxation in rabbit aorta after 30 min exposure to AGE-Glu is fully reversible and we found no evidence of short-term toxicity to HUVEC (up to at least 24 h incubation), although we observed a reduction in viable HUVEC after longer term (72 h) exposure to AGE-Glu.
Our data also suggest that AGE-Glu exerts its effects not through increased scavenging of NO, but through a decrease in its generation. The cell-permeable superoxide dismutase mimetic agent MnTMPyP did not prevent the inhibitory effect of AGE-Glu on ACh responses in rat aorta, nor were NP responses affected by AGE-Glu in this tissue. However, histamine-induced NOS activation in HUVEC was abolished by AGE-Glu, an effect again blocked by antibody to CML and by antibody to the AGE-R1 receptor. eNOS isolated from HUVEC exposed to AGE-Glu for 30 min exhibited a decrease in phosphoserine content, whereas eNOS expression was unchanged; the enzymic activity was abolished in eNOS isolated from HUVEC exposed to AGE-Glu. On the basis of our results, we suggest that AGE-Glu reversibly inhibits vascular NO generation through binding of CML residues to endothelial AGE-R1, which in turn causes a decrease in phosphorylation of serine residues in eNOS and thereby deactivation of the enzyme (see Fig. 3
). In the longer term, AGE-Glu may be cytotoxic to endothelial cells and, as described by others, may cause down-regulation of eNOS expression. The effects described here may play an important role in the pathogenesis of the endothelial dysfunction, and hence in the vascular disease, seen in diabetes mellitus.
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The precise mechanism by which AGE-R1 activation may decrease serine phosphorylation of eNOS remains to be determined. It may cause a decrease in protein kinase A- or Akt-mediated eNOS phosphorylation, both of which are known to modulate eNOS activity. Alternatively, it may increase the activity of one or more phosphatases, leading to increased dephosphorylation of eNOS. Further work is needed to distinguish between these possible mechanisms.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0490fje; doi: 10.1096/fj.02-0490fje 
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