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Angiogenesis induced by advanced glycation end products and its prevention by cerivastatin ¹

TAMAMI OKAMOTO^*, SHO-ICHI YAMAGISHI^*2, YOSUKE INAGAKI^*, SHINJIRO AMANO^*, KOHACHIRO KOGA^*, RIICHIRO ABE^¶, MASAYOSHI TAKEUCHI, SHIGEAKI OHNO, AKIHIKO YOSHIMURA^# and ZENJI MAKITA^*

^* Division of Endocrinology and Metabolism, Kurume University School of Medicine, Kurume 830-0011, Japan;
^¶ Department of Dermatology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan;
Department of Biochemistry, Hokuriku University, Kanazawa 920-1181, Japan;
Department of Ophthalmology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan; and
^# Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan

²Correspondence: Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. E-mail: shoichi{at}med.kurume-u.ac.jp

SPECIFIC AIMS

We investigated whether advanced glycation end products (AGE), which are senescent macroproteins that form at an accelerated rate in diabetes, elicit changes in cultured endothelial cells (EC) associated with angiogenesis. Angiogenesis is an important characteristic of the vascular derangements in diabetic retinopathy; therefore, we further examined whether cerivastatin, a hydroxymethylglutaryl CoA reductase inhibitor, could prevent AGE-induced angiogenesis by blocking the intracellular signaling pathways of AGE in human microvascular EC.

PRINCIPAL FINDINGS

1. AGE increased DNA synthesis and tube formation in microvascular EC through interaction with a receptor for AGE (RAGE)
We prepared various AGE proteins in vitro by incubating bovine serum albumin (BSA) with D-glyceraldehyde, glycolaldehyde, methylglyoxal, or glyoxal. Of the various AGE proteins, glyceraldehyde- and glycolaldehyde-derived AGE (glycer- and glycol-AGE) were found to significantly stimulate DNA synthesis as well as tube formation in cultured microvascular EC. Since we had previously found that glycer- or glycol-AGE were representative ligands for RAGE, we investigated whether the effects of these AGE would be mediated through interaction with RAGE. RAGE overexpression was found to significantly potentiate the AGE-induced increase in DNA synthesis in microvascular EC.

2. AGE up-regulated mRNA levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in microvascular EC
We have shown before that glucose-derived AGE elicit angiogenesis through overproduction of autocrine VEGF. Accordingly, we investigated whether glycer- or glycol-AGE induced expression of angiogenesis-related genes such as VEGF, kinase insert domain-containing receptor (kdr), Ang-1, Ang-2, or Tie-2 in microvascular EC. Glycer- and glycol-AGE significantly up-regulated mRNA levels of VEGF and Ang-2 but not kdr, Ang-1, or Tie-2 in EC. We confirmed that AGE stimulated the secretion of VEGF proteins by EC.

3. AGE induced promoter activity of nuclear factor-B (NF-B) and activation protein-1 (AP-1) and the resultant up-regulation of VEGF mRNA in microvascular EC
To elucidate the intracellular signaling pathways of AGE–RAGE systems in microvascular EC, we examined whether glycer- and glycol-AGE could stimulate the transcriptional activation of transcription factors NF-B and AP-1. Glycer- or glycol-AGE were found to significantly increase the transcriptional activity of NF-B and AP-1 in microvascular EC. Pyrrolidinedithiocarbamate and curcumin, inhibitors of the transcription factors NF-B and AP-1, respectively, completely prevented the AGE-induced up-regulation of VEGF mRNAs and the subsequent increase in DNA synthesis in EC. The results suggested that NF-B and AP-1 activation might be involved in the AGE-elicited angiogenesis through overproduction of autocrine VEGF proteins.

4. Cerivastatin prevented AGE-induced angiogenesis by blocking intracellular signaling pathways of AGE in microvascular EC
Cerivastatin, a potent inhibitor of cholesterol biosynthesis, has been known to have pleiotropic properties by blocking synthesis of isoprenoid intermediates, which serve as lipid attachments for a variety of intracellular signaling molecules. Therefore, we investigated whether cerivastatin could prevent AGE-elicited angiogenesis. As shown in Fig. 1 , cerivastatin was found to prevent AGE-induced up-regulation of VEGF mRNAs by blocking the transcriptional activation of NF-B and AP-1, resulting in the inhibition of angiogenesis elicited by AGE. Anti-angiogenic effect of cerivastatin was reversed by 100 µM mevalonate. A farnesyltransferase inhibitor (FTI-276) but not a geranylgeranyl transferase inhibitor (GGTI-286) was also found to inhibit AGE-induced increase in VEGF mRNA levels and the DNA synthesis in EC. These results suggest that the small G-protein Ras rather than Rho might be involved in AGE signaling pathways in microvascular EC.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effects of cerivastatin on AGE-induced transcriptional activation of NF-B and AP-1 (A), up-regulation of VEGF mRNA (B), increase in DNA synthesis (C), and tube formation (D) in microvascular EC. EC were incubated with 100 µg/mL of glycer-, glycol-AGE, or nonglycated BSA in the presence or absence of 5 nM cerivastatin or 100 µM mevalonate for 24 h. A) NF-B and AP-1 promoter activities. *P < 0.01 compared to value of the control of nonglycated BSA alone. #P < 0.05 compared to value of the control without cerivastatin. Black bars, values with 5 nM cerivastatin. B) RT-PCR analysis. Lower panel shows quantitative representation of VEGF gene induction. *P < 0.01, #P < 0.05 compared to value of the control with nonglycated BSA alone. C) [3H]Thymidine incorporation. *P < 0.01 compared to value of the control with nonglycated BSA. #P < 0.01 compared to value with cerivastatin alone. Black bars, values with 5 nM cerivastatin alone. Striped bars, values with 5 nM cerivastatin plus 100 µM mevalonate. D) Tube formation. *P < 0.01 compared to value of the control of nonglycated BSA alone. #P < 0.01 compared to value of the control without cerivastatin. Black bars, values with 5 nM cerivastatin.

CONCLUSIONS

Here we demonstrate for the first time that glycer- or glycol-AGE up-regulate VEGF mRNA levels and stimulate DNA synthesis and tube formation in microvascular EC through interaction with RAGE. This study has extended our previous work showing that glucose-derived AGE elicit angiogenesis by overproduction of autocrine VEGF proteins in EC. We recently showed that the structural epitope of these in vitro-modified AGE proteins exist in the serum of diabetic patients. These AGE proteins elicit angiogenesis at concentrations present in the plasma of diabetic patients. The results therefore suggest the relevance of these AGE epitopes in the pathological angiogenesis in vivo.

Microvessels are composed of two types of cells: EC and pericytes. Pericytes not only regulate the growth but also preserve the prostacyclin-producing ability of cocultured EC, thus playing a central role in the maintenance of microvascular hemostasis. We recently found that glycer- or glycol-AGE induced apoptotic cell death of retinal pericytes through interaction with RAGE. The AGE were found to simultaneously stimulate VEGF production in pericytes. The results suggest that the AGE–RAGE interaction could facilitate angiogenesis by two distinct mechanisms: one is by the relief of restriction on EC growth by apoptotic cell death of pericytes, the other is by an autocrine and paracrine induction of VEGF proteins by vascular wall cells.

We have found for the first time that AGE significantly up-regulated Ang-2 but not Ang-1 mRNA levels in microvascular EC. Ang-2, a naturally occurring antagonist of Ang-1 that competes for tyrosine kinase receptor Tie-2, has been known to induce the loosening of contact between microvascular EC and pericytes. Since a plastic window for vessel remodeling is defined by pericyte coverage of the preformed endothelial network, AGE-induced pericyte loss and Ang-2 induction could both elicit angiogenesis in concert with VEGF, contributing to the pathogenesis of proliferative diabetic retinopathy.

We showed in this study that AGE up-regulated VEGF mRNA levels through the transcription factors NF-B and AP-1 in microvascular EC. Angiotensin II has been found to induce VEGF mRNA levels through transcriptional activation of NF-B and AP-1 in heart-derived EC. Since reactive oxygen species (ROS) are known to be involved in angiotensin II signaling, AGE–RAGE interaction and the ROS generation would stimulate VEGF mRNA induction through these redox-sensitive transcription factors.

We found that cerivastatin prevented AGE-elicited angiogenesis by inhibiting the intracellular AGE signaling pathways in microvascular EC. Cerivastatin has been shown to block synthesis of isoprenoid intermediates, which are essential for membrane attachment and biological activity of the small G-proteins such as Ras and Rho. In this study, a farnesyl transferase inhibitor (FTI-276) but not a geranylgeranyl transferase inhibitor (GGTI-286) was found to prevent AGE-induced VEGF mRNA up-regulation and the resultant growth stimulation of EC. These results suggest that Ras rather than Rho protein is required for the AGE–RAGE signaling pathways in microvascular EC. The AGE–RAGE-induced ROS generation and subsequent Ras activation may lead to VEGF mRNA up-regulation through transcriptional activation of NF-B and AP-1. Ras has been reported to activate transcription factors NF-B and AP-1 through mitogen-activated protein kinase in EC, supporting our speculation (Fig. 2 ).

View larger version (34K): [in this window] [in a new window]   Figure 2. Possible mechanism of the AGE-induced angiogenesis and its prevention.

Our study suggests that cerivastatin may be a promising remedy for treatment of patients with proliferative diabetic retinopathy by interfering with the intracellular AGE–RAGE signaling pathways in microvascular EC.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0030fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0030fje

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