Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

https://doi.org/10.1016/j.bbrc.2009.02.040Get rights and content

Abstract

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.

Section snippets

Materials and methods

Synthesis of AGE-modified albumin. AGE-modified albumin (AGE-albumin) was synthesized under sterile conditions by incubating 20 mg/ml bovine serum albumin (BSA, low endotoxin, Merck) with 50 mmol/L glucose for 90 days, following which the mixture was fully dialyzed against PBS to remove unbound glucose, as previously described [7]. BSA incubated without glucose under the same conditions was used as the negative control in all experiments.

EPC isolation and culture. The study followed procedures in

Characterization of EPCs

Two types of cells were observed in our culture system. One type (“early EPCs”), appeared 3–5 days after primary culture. These early EPCs were spindle shaped and appeared as clusters, but these clusters disappeared in 10–14 days. Another cell population (“late EPCs”) appeared as clusters 10–15 days after plating. Cells (20–50) with smooth cytoplasmic outlines were observed in each cluster, and each cluster gave rise to more than 500 cells, which grew to confluence and exhibited a cobblestone

Discussion

The range of AGE-albumin concentrations used in the present study (50–400 μg/ml) was chosen because it is representative of plasma concentrations of AGE-albumin found in diabetic patients, as we have previously reported [7]. Therefore, the effects of AGE-albumin observed here are likely to be replicated in diabetic patients in vivo.

Sun et al. [20] have previously reported that, when EPCs derived from peripheral BMCs are incubated with 200 μg/ml AGE-albumin for 24 h, decreased proliferation and

Acknowledgments

This project was supported by the National Science Foundation of China (Grant No. 03030201) and the Natural Science Foundation of Jiangsu Province of China (Grant No. BK2004083).

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