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*

(Received for publication, August 29, 1996, and in revised form, December 23, 1996)

Sho-ichi Yamagishi Dagger , Hideto Yonekura Dagger , Yasuhiko Yamamoto Dagger , Kenji Katsuno §, Fumiyasu Sato §, Izumi Mita , Hisayoshi Ooka , Noboru Satozawa par , Takuhisa Kawakami Dagger , Motohiro Nomura Dagger and Hiroshi Yamamoto Dagger **

From the Dagger  Department of Biochemistry, Kanazawa University School of Medicine, Kanazawa 920, Japan, the § Discovery Research Laboratory 3, Kissei Pharmaceutical Co. Ltd., Hotaka 399-83, Japan, the  Institute of Biological Science, Mitsui Pharmaceuticals Inc, Mobara 297, Japan, and the par  Life Science Laboratory, Mitsui Toatsu Chemicals Inc., Mobara 297, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

This study was undertaken to determine whether and how advanced glycation end products (AGE), senescent macroproteins accumulated in various tissues under hyperglycemic states, cause angiogenesis, the principal vascular derangement in diabetic microangiopathy. We first prepared AGE-bovine serum albumin (BSA) and anti-AGE antiserum using AGE-RNase A. Then AGE-BSA was administered to human skin microvascular endothelial cells in culture, and their growth was examined. The AGE-BSA, but not nonglycated BSA, was found to induce a statistically significant increase in the number of viable endothelial cells as well as their synthesis of DNA. The increase in DNA synthesis by AGE-BSA was abolished by anti-AGE antibodies. AGE-BSA also stimulated the tube formation of endothelial cells on Matrigel. We obtained the following evidence that it is vascular endothelial growth factor (VEGF) that mainly mediates the angiogenic activities of AGE. (1) Quantitative reverse transcription-polymerase chain reaction analysis of poly(A)+ RNA from microvascular endothelial cells revealed that AGE-BSA up-regulated the levels of mRNAs for the secretory forms of VEGF in time- and dose-dependent manners, while endothelial cell expression of the genes encoding the two VEGF receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase 1, remained unchanged by the AGE treatment. Immunoprecipitation analysis revealed that AGE-BSA did increase de novo synthesis of VEGF. (2) Monoclonal antibody against human VEGF completely neutralized both the AGE-induced DNA synthesis and tube formation of the endothelial cells. The results suggest that AGE can elicit angiogenesis through the induction of autocrine vascular VEGF, thereby playing an active part in the development and progression of diabetic microangiopathies.


INTRODUCTION

Glucose and other reducing sugars can react nonenzymatically with the amino groups of proteins to form reversible Schiff bases and, then, Amadori products. These early glycation products undergo further complex reactions such as rearrangement, dehydration, and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives termed advanced glycation end products (AGE)1 (1). The formation and accumulation of AGE in various tissues have been known to progress during normal aging and at an extremely accelerated rate in diabetes mellitus. This has been implicated in the development of diabetic micro- and macro-vascular complications (1), which may account for the disabilities and high mortality rate in patients with this disease (2).

Microvessels are composed of only two types of cells, endothelial cells and pericytes, and have been known to show both functional and structural abnormalities during prolonged diabetic exposure, resulting in the deleterious effects on the organs that they supply (3-5). Using pericyte-endothelial cell co-culture systems, we have shown previously that pericytes can not only regulate the growth but also preserve the prostacyclin-producing ability and protect against lipid peroxide-induced injury of endothelial cells (6). This has provided a basis for understanding how diabetic retinopathy develops consequent to "pericyte loss," the earliest histopathological hallmark in diabetic retinopathy (5, 7).

Recently, we have found that AGE exert a growth inhibitory effect and a cell type-specific immediate toxicity on pericytes through interactions with their receptor for AGE (RAGE), a cell surface receptor belonging to the immunoglobulin superfamily (8), and have proposed a novel mechanism for pericyte loss (9). The AGE-induced, RAGE-mediated decrease in pericyte number would then indirectly cause angiogenesis (6, 9).

In the present study, we investigated the effects of AGE on the growth and tube formation of human skin microvascular endothelial cells, the key steps of angiogenesis. We demonstrate that AGE exert angiogenic activities directly on microvascular endothelial cells and that autocrine vascular endothelial growth factor (VEGF) is the major mediator of the AGE-driven angiogenesis.


EXPERIMENTAL PROCEDURES

Materials

Bovine serum albumin (BSA) was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Bovine pancreatic RNase A, bovine hemoglobin (Hb), Nalpha -tosyl-lysine methyl ester and phenylmethylsulfonyl fluoride were from Sigma. Heparin-Sepharose CL-4B was from Pharmacia LKB (Uppsala, Sweden). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was from BioMakor (Rehovot, Israel). [3H]Thymidine and [gamma -32P]ATP were from DuPont NEN. Reverse transcriptase and T4 polynucleotide kinase were from Takara (Kyoto, Japan). Hybond-N+ nylon membrane was from Amersham Corp. (Buckinghamshire, United Kingdom). Matrigel was from Collaborative Research (Bedford, MA).

Preparation of AGE-Proteins and Amadori Compounds

BSA (fraction V, fatty acid-free, free endotoxin) was incubated with 0.5 M glucose at 37 °C for 6 weeks under sterile conditions in the presence of 1.5 mM phenylmethylsulfonyl fluoride, 0.5 mM EDTA, 100 units/ml penicillin, and 40 µg/ml gentamycin (9-11). After unincorporated sugars were removed by dialysis against phosphate-buffered saline, glucose-modified high molecular weight materials were purified by heparin-Sepharose CL-4B column chromatography and used as AGE-BSA. Control nonglycated BSA was incubated in the same conditions except for the absence of glucose. The concentration of AGE-BSA was determined by the method of Bradford (12). AGE-RNase A and AGE-Hb were prepared according to the method of Makita et al. (13). For reducing AGE-Hb, NaBH4 was employed as described by Horiuchi et al. (14). 1-Deoxy-1-propylamino-D-fructose, an Amadori compound, was synthesized by the method of Micheel and Hagemann (15). Nalpha -Tosyl-Nepsilon -carboxymethyllysine methyl ester (CM-TsLME) was synthesized from Nalpha -tosyl-lysine methyl ester by the method of Ahmed et al. (16) with minor modifications. N-Carboxymethylated BSA (CM-BSA) was prepared according to the method of Reddy et al. (17).

Preparation of Anti-AGE-RNase A Antiserum

1 mg of AGE-RNase A was emulsified in 50% Freund complete adjuvant and injected intradermally into rabbits. Two weeks later, a booster with the same amount of AGE-RNase A was administered, followed by nine additional booster injections, with one given every 2-3 weeks. Ten days after the final injection, the antiserum was obtained.

Enzyme-linked Immunosorbent Assay (ELISA)

In the noncompetitive ELISA system, wells of 96-well microtiter plates were coated with increasing amounts of Hb, AGE-Hb, and reduced AGE-Hb. After washing and blocking, the wells were incubated with 100 µl of anti-AGE antiserum (1:4000) for 2 h and then with 100 µl of HRP-conjugated goat anti-rabbit IgG (1:2000) for 30 min. Finally, 100 µl of substrate tetramethylbenzidine solution was added into each well. After 10-15 min, the absorbance at 450 nm was measured. In the competitive ELISA system, procedures similar to the noncompetitive ELISA were used except for the following two points. Wells were first coated with reduced 100 ng/ml AGE-Hb solution as absorbent antigens, and then 50 µl of test samples were added as a competitor together with 50 µl of anti-AGE antiserum (1:2000) into each well.

Cells

Endothelial cells from human skin microvessels were maintained in E-BM medium supplemented with 5% fetal bovine serum, 0.4% bovine brain extracts, 10 ng/ml human epidermal growth factor, and 1 µg/ml hydrocortisone according to the supplier instructions (Clonetics Corp., San Diego, CA). Cells at 5-10 passages were used for the experiments. AGE treatment was carried out in a medium lacking epidermal growth factor and hydrocortisone.

Measurement of Cell Growth

Endothelial cells cultured for various time periods in the presence or absence of AGE-BSA were dislodged with trypsin, and counted by the dye exclusion method (18). [3H]Thymidine incorporation was determined as described previously (19). For determining the effects of anti-AGE antiserum on endothelial cell growth, the antiserum was added to the medium at 1% (v/v) together with or without 50 µg/ml AGE-BSA, after which cells were incubated for 24 h and [3H]thymidine incorporation was measured.

Primers and Probes

Oligonucleotide primers and probes for quantitative reverse transcription-polymerase chain reactions (RT-PCR) were synthesized by a Perkin-Elmer 392 DNA synthesizer (Foster City, CA) and purified as described previously (20). Primer sequences and internal oligonucleotide probes for detecting VEGF, fms-like tyrosine kinase 1 (flt 1), kinase insert domain-containing receptor (kdr), and beta -actin mRNA were the same as described in Ref. 20.

Quantitative RT-PCR

Poly(A)+ RNAs were isolated (21) from cells treated with or without AGE-BSA for the various time periods and analyzed by RT-PCR as described previously (22). 6-µl aliquots of each RT-PCR reaction mixture were electrophoresed on a 2% agarose gel and transferred to a Hybond-N+ nylon membrane, and the membrane was hybridized with the respective 32P-end-labeled probes (20). The amounts of poly(A)+ RNA templates (30 ng) and cycle numbers (30 cycles) for amplification were chosen in quantitative ranges where reactions proceeded linearly, which had been determined by plotting signal intensities as functions of the template amounts and cycle numbers (20). Signal intensities of hybridized bands were measured by a Fujix BAS 1000 Image analyzer (Fuji Photo Film Co. Ltd., Hamamatsu, Japan).

Metabolic Labeling and Immunoprecipitation of VEGF

Subconfluent cultures of endothelial cells were incubated in the presence or absence of 50 µg/ml AGE-BSA for 2 h and further incubated for 6 h at 37 °C in methionine-free RPMI 1640 medium/complete RPMI 1640 medium (9:1) containing 0.2 mCi/ml [35S]methionine with or without AGE-BSA. Immunoprecipitation was carried out as described previously (20). The samples were analyzed on a 15% SDS-polyacrylamide gel under reducing conditions.

Preparation of Monoclonal Antibody (MoAb) against Human VEGF

Peripheral blood lymphocytes from healthy volunteers were immortalized by Epstein-Barr virus, and the resultant Epstein-Barr virus-transformants were screened (23) for their ability to produce antibody molecules that bind to recombinant human VEGF165 (Peprotech, Rocky Hill, NJ). The candidate cells were then fused with SHM-D33 human myeloma cells (American Type Culture Collection, Rockville, MD). A cell line that consistently produced an IgM-MoAb reactive to VEGF was cloned and designated BL-2. Then the BL-2 MoAb was purified from the serum-free supernatant by 50% ammonium sulfate precipitation and by Sephadex G-200 gel chromatography. The specific binding of MoAb BL-2 to VEGF was confirmed by immunoblotting.2

Tube Formation in Vitro

Wells of 24-well culture cluster dishes (Coster 3524, Cambridge, MA) were coated with Matrigel solution (250 µl/well) and then allowed to solidify for at least 1 h at 37 °C (24, 25). Endothelial cells (4 × 104 cells/well) were then seeded on Matrigel with or without 10 µg/ml MoAb BL-2. After 30 min, the cells were treated with or without 50 µg/ml AGE-BSA for 6 h, 4 microscopic fields selected at random were photographed, and the lengths of tube-like structures were measured with microcomputer-assisted NIH Image (Version 1.56).


RESULTS

Characterization of AGE-BSA

AGE-BSA was prepared by incubating BSA with glucose and then purified by heparin-Sepharose CL-4B column chromatography. Fig. 1 shows its electrophoretic profile on reducing SDS-polyacrylamide gel electrophoresis. Control nonglycated BSA migrated to the position at 68 kDa. On the other hand, the purified materials migrated much more slowly, yielding a broad band larger than 68 kDa. This indicated that covalently linked adducts were formed nonenzymatically on BSA without discernible degradation. As shown in Table I, the purified materials also exhibited spectrophotometric features characteristic to AGE (26, 27). The peak fluorescence was noted at 440 nm with excitation at 370 nm, and its intensity was increased 10-fold in comparison with nonglycated BSA. Chromogen products also appeared in the purified materials, whereas they were barely detectable in nonglycated BSA. Based on these observations, the purified materials were used as AGE-BSA.


Fig. 1. SDS-PAGE of AGE-BSA. 10 µg of nonglycated native BSA (lane 1) and AGE-BSA (lane 2) were loaded on a 10% polyacrylamide gel with a stacking gel of 5% polyacrylamide. Staining of the gel was performed with Coomassie Brilliant Blue. Size markers (kDa) are shown on the left.
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Table I.

Characterization of AGE-BSA

Data represent the mean ± S.E. of three replicate experiments. All the samples were adjusted to a protein concentration of 1.0 mg/ml.


Fluorescencea Chromogen productsb

arbitrary units
BSA 1.0  ± 0.0 0.0  ± 0.0
AGE-BSA 9.9  ± 0.1 0.11  ± 0.0

a An arbitrary value of 1 was assigned to fluorescence of control BSA. Fluorescence was measured at excitation of 370 nm and emission of 440 nm.
b Measured as absorbance at 350 nm/absorbance at 280 nm.

Characterization of Anti-AGE Antiserum

As a tool to evaluate AGE and their biological effects, an antiserum was raised against AGE-RNase A. The reactivity of this antiserum to several AGE-modified proteins, Amadori compounds, and carboxymethyllysine derivatives was examined. Since it was possible that AGE-modified proteins might contain early glycation products, the immunoreactivity of the antiserum to reduced AGE-Hb was also tested because the early glycation products are known to be converted to glucitol-lysine by reduction with NaBH4 (14, 28). As shown in Fig. 2A, there was no difference in the immunoreactivity in the noncompetitive ELISA between AGE-Hb and reduced AGE-Hb. Further, as shown in Fig. 2B, the antiserum binding to AGE-Hb was not competed for by 1-deoxy-1-propylamino-D-fructose in the competitive ELISA, indicating that the antiserum does not recognize Amadori compounds. We next tested whether the antiserum reacted to CM-TsLME and CM-BSA in the competitive ELISA. As shown in Fig. 2, B and C, these glycoxidative products were found to partially inhibit the antiserum binding to reduced AGE-Hb, whereas AGE-Hb and AGE-RNase A fully inhibited its binding (Fig. 2D). Further, the antiserum reactivity to AGE-BSA was dependent on the duration of incubation of BSA with glucose (Fig. 2E). These results suggested that the antiserum could recongnize AGE structures common to various AGE preparations.


Fig. 2. Characterization of anti-AGE antiserum. A, antiserum was titered in noncompetitive ELISA using Hb (open circle ), AGE-Hb (bullet ), and reduced AGE-Hb (black-triangle) as absorbent antigens, as described under "Experimental Procedures." The amounts of the antigens are indicated on the abscissa, and absorbance at 450 nm is on the ordinate. B-D, antiserum was titered in competitive ELISA. Wells were coated with reduced AGE-Hb and then various test samples as competitor, and anti-AGE antiserum were added. B, 1-deoxy-1-propylamino-D-fructose (bullet ); CM-TsLME (open circle ). C, CM-BSA (bullet ); 8-week incubated AGE-BSA (open circle ). D, Hb (open circle ); 8-week incubated AGE-Hb (bullet ); 8-week incubated AGE-RNase A (black-triangle). E, BSA (open circle ); 4-week incubated AGE-BSA (bullet ); 8-week incubated AGE-BSA (black-triangle); 12-week incubated AGE-BSA (black-square). The amounts of the competitors are indicated on the abscissa, and absorbance at 450 nm is on the ordinate. Similar results were obtained in two independent experiments.
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Stimulation of the Growth of Microvascular Endothelial Cells by AGE

Endothelial cells obtained from human skin microvessels were cultured in the presence or absence of AGE-BSA, and the viable cell number was determined at days 1, 2, and 3 after the AGE addition. As shown in Fig. 3, AGE-BSA was found to increase the viable microvascular endothelial cell number in a dose-dependent manner; at 50 µg/ml AGE, there was about a 40% increase in viable cell number. Moreover, AGE-BSA significantly increased DNA synthesis in microvascular endothelial cells to 130% (p < 0.01, Fig. 4). However, nonglycated BSA induced no change in either the cell number or DNA synthesis.


Fig. 3. Effects of AGE-BSA on viable cell number of human skin microvascular endothelial cells. 1 × 104 endothelial cells were seeded per well and grown in the presence of 1 (bullet ), 10 (square ), or 50 (black-square) µg/ml AGE-BSA or in the absence of AGE-BSA (open circle ). The culture period after the addition of AGE-BSA is indicated on the abscissa, and the viable cell number is on the ordinate. Each point represents the mean ± S.E. of three replicate experiments. *, p < 0.05; **, p < 0.01, compared with the control value without additives (Student's t test).
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Fig. 4. Effects of antiserum against AGE-RNase A on the AGE-induced DNA synthesis in human skin microvascular endothelial cells. Endothelial cells were treated with antiserum against AGE-RNase A in the presence or absence of 50 µg/ml AGE-BSA. After incubation for 24 h, [3H]thymidine was added, and then its incorporation into the cells was assayed. The percentage of [3H]thymidine incorporation is indicated on the ordinate and related to the value for the control with no additives. Each column represents the mean ± S.E. of four replicate experiments. *, p < 0.05; **, p < 0.01, compared with the value with 50 µg/ml AGE-BSA alone (Student's t test).
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Neutralization of the AGE-induced DNA Synthesis by Anti-AGE Antibody

To evaluate the specificity of the AGE-BSA effect on endothelial cell growth, we examined the effects of the antiserum against AGE-RNase A on AGE-induced DNA synthesis. As shown in Fig. 4, the anti-AGE-RNase A antiserum was found to completely neutralize the AGE-induced synthesis of endothelial cell DNA at 1%, while the same concentration of the antiserum did not affect DNA synthesis in endothelial cells not exposed to AGE-BSA.

Microvascular Endothelial Cells Express mRNA for Secretory Forms of VEGF in Response to AGE

Poly(A)+ RNAs were isolated from microvascular endothelial cells treated with various concentrations of AGE-BSA for various time periods, and analyzed by a quantitative RT-PCR technique to determine the effects of AGE on the expression of the VEGF gene. It has been reported that there are four alternatively spliced products from the single VEGF gene, VEGF121, VEGF165, VEGF189, and VEGF206 (29, 30). Since Northern blot analysis cannot clearly discriminate the four mRNA species, we employed a more sensitive RT-PCR technique as described previously (20). In this experiment, 486- and 618-base pairs (bp)-long cDNA products would be amplified from mRNAs for VEGF121 and VEGF165, respectively (20).

As shown in Fig. 5, A and B, microvascular endothelial cells were expressing mRNAs for VEGF121 and VEGF165, the secretory forms of VEGF. When the endothelial cells were exposed to AGE-BSA, the level of VEGF mRNAs was found to be significantly increased in a time- and dose-dependent manner. The VEGF mRNA level began to increase at 2 h, and reached a maximum at 4 h in the presence of 50 µg/ml AGE-BSA; the peak value was 3-fold higher than the basal level when standardized with the signal intensities of beta -actin mRNA as an internal control. Maximal stimulation was achieved at 50-100 µg/ml AGE. However, the larger alternatively spliced products coding for VEGF189 and VEGF206 were not detected in microvascular endothelial cells regardless of the presence or absence of AGE-BSA.


Fig. 5. Expression of VEGF and its receptor genes in microvascular endothelial cells. A, time course of VEGF mRNA induction by AGE. Endothelial cells were treated with 50 µg/ml AGE, and then 30 ng of poly(A)+ RNAs were transcribed and amplified by PCR at the indicated times. B, dose-response of VEGF mRNA induction by AGE. Endothelial cells were incubated for 4 h with the indicated concentrations of AGE, and then 30 ng of poly(A)+ RNAs were amplified by RT-PCR. Lower panels show expression of VEGF receptor genes. 30 ng of poly(A)+ RNAs from endothelial cells treated with the various concentrations of AGE were reverse transcribed, and PCR was performed for 25 cycles. The PCR products were electrophoresed on 2% agarose gel, transferred onto nylon membranes, and hybridized with 32P-end-labeled probes. PCR amplification for beta -actin mRNA was performed for 20 cycles. Size markers (bp) are shown on the left. C, VEGF synthesis in AGE-treated and untreated endothelial cells. Immunoprecipitation was carried out as described under "Experimental Procedures." Immunoreacted materials that had been prepared from 8.0 × 106 dpm of lysates were electrophoresed on a 15% SDS-polyacrylamide gel under reducing conditions. The gel was dried and autoradiographed. The radioactivities of the bands were measured with a Fujix BAS 1000 BioImage analyzer. Specific immunoprecipitates were marked at 22 and 18 kDa. Similar results were obtained in two independent experiments.
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To confirm whether AGE-BSA increased the synthesis of VEGF proteins, we performed immunoprecipitation using the anti-VEGF monoclonal antibody (20) from lysates of the cells that had been treated with or without AGE-BSA. As shown in Fig. 5C, 35S-labeled proteins that migrated to the positions of 18 and 22 kDa, corresponding to VEGF121 and VEGF165, respectively, were immunoprecipitated, and the amounts of these proteins were found to be increased to about 2.5-fold by the AGE treatment.

VEGF Receptor Expressions in Microvascular Endothelial Cells

VEGF exerts its biological actions through its specific receptors, KDR and Flt 1 (31, 32). We then determined the types of VEGF receptors expressed in microvascular endothelial cells and whether their expressions could be altered by AGE-BSA. As shown in Fig. 5B, both kdr and flt 1 mRNA were detected in microvascular endothelial cells, and the content of kdr mRNA was more abundant than that of flt 1 mRNA. In contrast to VEGF mRNAs, the levels of the two types of receptors were essentially unchanged by the exposure to AGE-BSA.

Neutralization of the AGE-Induced DNA Synthesis of Microvascular Endothelial Cells by MoAb against Human VEGF

We next investigated whether vascular VEGF may have a functional role in the AGE action on endothelial cells. Microvascular endothelial cells were preincubated with various concentrations of MoAb BL-2 for 30 min, then exposed to 50 µg/ml AGE-BSA for 24 h in the presence of the antibody, and assayed for [3H]thymidine incorporation. As shown in Fig. 6, MoAb BL-2 was found to significantly diminish the AGE-induced increase in DNA synthesis in a dose-dependent manner; at 10 µg/ml, a complete reversal was obtained. The MoAb alone did not affect DNA synthesis in endothelial cells not exposed to AGE.


Fig. 6. Effects of antibody against VEGF (BL-2) on the AGE-induced DNA synthesis. Endothelial cells were preincubated with the various concentrations of BL-2 for 30 min and then treated with or without 50 µg/ml AGE for 24 h. The percentage of [3H]thymidine incorporation is indicated on the ordinate. Each column represents the mean values of four replicate experiments. Bars show S.E. *, p < 0.05, compared with the values obtained in the presence of AGE alone (Student's t test).
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AGE Induction of Tube Formation of Microvascular Endothelial Cells and Its Inhibition by Anti-VEGF MoAb

The process of angiogenesis has been assumed to be completed by the formation of microvascular tubes (19). In vitro assays for tube formation of endothelial cells have been developed and used to study this crucial step of angiogenesis. Accordingly, we examined whether AGE affect in vitro tube formation of microvascular endothelial cells. For this, we employed an on-gel assay system using Matrigel, in which endothelial cells take only several hours to associate with each together and form microtubes. Microvascular endothelial cells were seeded on Matrigel with or without AGE-BSA, and tube formations were judged after 6 h. As shown in Fig. 7A, AGE-BSA was found to double the length of the tubes of endothelial cells formed on Matrigel; and, the AGE-induced tube formation was inhibited by BL-2 MoAb as was the AGE-induced DNA synthesis of endothelial cells. The MoAb per se did not affect the tube formation. Fig. 7B shows typical micrographs; with 10 µg/ml BL-2 MoAb, AGE-induced tube formation was markedly inhibited.


Fig. 7. Effects of antibody against VEGF (BL-2) on the AGE-induced tube formation in endothelial cells. A, endothelial cells were treated with or without 10 µg/ml antibody and then incubated in the presence or absence of 50 µg/ml AGE for 6 h. Each four fields selected at random were photographed, and the lengths of tube-like structures were quantitatively measured with microcomputer-assisted NIH Image. Each column represents the mean values of four replicate experiments. Bars show S.E. **, p < 0.01, compared with the values obtained in the presence of AGE alone (Student's t test). B, typical microphotographs of tube formations of endothelial cells. a, control without no additive; b, 10 µg/ml BL-2; c, 50 µg/ml AGE; d, 50 µg/ml AGE plus 10 µg/ml BL-2.
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DISCUSSION

In the present study, we have demonstrated for the first time that AGE, nonenzymatically glycated protein derivatives formed under hyperglycemia, stimulate the growth and tube formation of human microvascular endothelial cells, the key steps of angiogenesis which take place in this very cell type (33, 34). The present findings have extended our preliminary work employing endothelial cells from a larger vessel, i.e. the umbilical vein. Though modestly, AGE-BSA caused a consistent increase in cell number and in DNA synthesis of both umbilical and microvasular endothelial cells, the same AGE-BSA concentration (50 µg/ml) giving the maximal effect in both cases (35). This concentration of AGE was comparable with that of the in vivo situation in diabetes; Makita et al. reported (13) that human serum AGE levels were elevated more than 2-fold in diabetic patients (about 25 µg/ml) and almost 8-fold in diabetic patients on hemodialysis (about 80 µg/ml) in comparison with that in normal patients.

That it was AGE moieties that elicited the angiogenic activity was evidenced as follows. First, the AGE-BSA employed exhibited the biochemical hallmarks of AGE (Fig. 1 and Table I). Second, control nonglycated BSA made no change (data not shown). Third, a newly developed antiserum against AGE-RNase A could neutralize the growth-promoting effect of AGE-BSA (Fig. 4). Although this antiserum was partially reactive to carboxymethyllysine, it would seem unlikely that the AGE effect could be accounted for by such glycoxidative biproducts of the Maillard reaction, which might be present in trace amounts in the AGE-BSA preparation, because authentic carboxymethylated BSA added to the culture medium at concentrations from 1 to 100 µg/ml failed to stimulate the endothelial cell synthesis of DNA (data not shown). We speculate that the AGE actions on microvascular endothelial cells may require their binding to RAGE, the AGE-specific receptor, as is the case with bovine retinal pericytes (9), human umbilical vein endothelial cells (35), and human pancreatic cancer cells (36).

The present study has also demonstrated that the angiogenic activity of AGE is mainly mediated by autocrine VEGF synthesized by micrcovascular endothelial cells per se. mRNAs for VEGF121 and VEGF165 are present in microvascular endothelial cells, and their levels are up-regulated by AGE in both dose- and time-dependent manners (Fig. 5, A and B). AGE did increase de novo synthesis of VEGF in endothelial cells (Fig. 5C). mRNAs for the two VEGF receptors, kdr and flt 1, were also detected in human skin microvascular endothelial cells, their relative abundance being kdr >>  flt 1. However, their levels were essentially unaltered when exposed to AGE (Fig. 5B). This suggests that the ligand expression should be the rate-limiting step in the putative autocrine action of VEGF. In effect, the neutralization experiments established the functional role of VEGF in the AGE-induced endothelial cell growth and tube formation. MoAb against human VEGF could completely inhibit the AGE-induced tube formation as well as the DNA synthesis of microvascular endothelial cells (Figs. 6 and 7). Since the basal growth or tube formation in unexposed cells was not affected by the same concentration of the antibody, the antibody-induced inhibition is not likely the result of its toxic or nonspecific effects. These results thus indicate that autocrine VEGF is the main mediator of the AGE-driven angiogenesis in vitro.

In light of the present findings, together with the previous observations, we can now posit an overall scheme concerning the roles of AGE in the development of diabetic microangiopathy (Fig. 8). First, AGE act on pericytes, the microvascular constituent that encircles the endothelium. Through interactions with RAGE, AGE decrease the number of this cell type (9), leading to pericyte dropout, which would in turn relieve the restriction on endothelial cell replication and facilitate angiogenesis. The resultant cessation of pericyte-endothelial cell interactions would impair prostacyclin production (6), which would cause thrombogenesis. Second, AGE act on endothelial cells, which serve as a barrier between circulating blood and parenchyma and produced various vasoactive substances. As shown in this paper, one consequence is angiogenesis that is probably mediated by autocrine VEGF. In addition, the prostacyclin-synthesizing ability of microvascular endothelial cells might be directly inhibited by AGE, as in umbilical endothelial cells (35). Again, the consequence of this would be thrombogenesis. Diminished circulation or microthrombus formation may occur in such lesions, giving rise to hypoxia, the major factor triggering VEGF expression in both endothelial cells and pericytes (20, 37-40). In such circumstances, angiogenesis would further proceed, which may eventually lead to the clinical expression of diabetic microangiopathies, exemplified by proliferative retinopathy. According to this model, procedures that can halt those events, e.g. inhibition of AGE formation,3 AGE absorption by immobilized antibodies, antisense RAGE DNA (35, 36), prostacyclin analogues, or anti-VEGF neutralizing antibodies would then theoretically help circumvent the development and progression of diabetic microangiopathies.


Fig. 8. Possible mechanism of the development of diabetic microangiopathy. AGE, advanced glycation end products; RAGE, a receptor for AGE; VEGF, vascular endothelial growth factor. Concerning (1) pericyte-endothelial cell interactions, (2) AGE effect on pericytes, (3) AGE-RAGE interaction in umbilical vein endothelial cells, and (4) hypoxia induction of vascular VEGF, refer to (1) Refs. 6 and 18, (2) Ref. 9, (3) Ref. 35, and (4) Ref. 20, respectively.
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Although this view basically stands on the in vitro experiments, in support are several in vivo or clinical observations. Hammes et al. (41, 42) reported that aminoguanidine, an inhibitor of AGE formation, prevented AGE accumulation at branching sites of precapillary arterioles and that this inhibitor could diminish pericyte dropout and inhibit abnormal endothelial cell proliferation in streptozotocin-induced diabetic rats. Dolhofer-Bliesener et al. (43) have shown that, in human diabetic subjects, the serum level of AGE was associated with the state of late complications, particularly in cases with retinopathy. Wautier et al. (44) reported that infusion of diabetic red blood cells into normal rats can induce vascular hyperpermeability, which was completely inhibited by anti-RAGE IgG; they also demonstrated that an antioxidant, probucol, can similarly reverse the red blood cell transfer-induced vascular permeability, suggesting a role of AGE-RAGE interactions and the involvement of an oxidant stress-sensitive pathway in the development of hyperpermeability. Recent clinical studies at different institutions have established that intraocular concentrations of VEGF correlated with active neovascularization (45, 46). Further, the increased permeability of retinal capillaries and the breakdown of the blood-retinal barrier have been shown to be as an early event in both human diabetic subjects and streptozotocin diabetic rats followed by new vessel formation (47, 48). The barrier breakdown would help ensure pericyte access to AGE. Moreover, it is reported that the barrier breakdown could be protected by the treatment of aminoguanidine, suggesting that AGE themselves may be directly involved in the barrier breakdown (48).


FOOTNOTES

*   This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan, the Sagawa Foundation for Promotion of Cancer Research, Japan, and the Japan Diabetes Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence and requests for reprints should be addressed. Tel.: 81-76-265-2180; Fax: 81-76-234-4226).
1   The abbreviations used are: AGE, advanced glycation end products; RAGE, receptor for AGE; VEGF, vascular endothelial growth factor; BSA, bovine serum albumin; Hb, hemoglobin; HRP, horseradish peroxidase; CM-TsLME, Nalpha -tosyl-Nepsilon -carboxymethyllysine methyl ester; CM-BSA, N-carboxymethylated BSA; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; Flt 1, fms-like tyrosine kinase 1; KDR, kinase insert domain-containing receptor; MoAb, monoclonal antibody; bp, base pairs.
2   The monoclonal antibody did not cross-react with basic fibroblast growth factor or platelet-derived growth factor. Its binding to VEGF-B (49) and -C (50), recently described members of the VEGF family, was undetectable and about 1/1000 of that to VEGF, respectively. VEGF-B and -C proteins were donated from the Ludwig Institute for Cancer Research and University of Helsinki.
3   Sato et al. (51) have recently developed a novel class of inhibitors of AGE formation, which are distinct from aminoguanidine in both structure and mode of action.

ACKNOWLEDGEMENTS

We thank Professor Kari Alitalo, Helsinki University and Dr. Ulf Eriksson, Ludwig Institute for Cancer Research for kindly providing VEGF-B and VEGF-C, Kurabo Industries, Ltd., Osaka for human capillary endothelial cells, Shin-ichi Matsudaira and Reiko Kitamura for assistance, and Brent Bell for reading the manuscript.


REFERENCES

  1. Brownlee, M., Cerami, A., and Vlassara, H. (1988) N. Engl. J. Med. 318, 1315-1321 [Medline] [Order article via Infotrieve]
  2. Krolewski, A., Warram, J., Valsania, P., Martin, B., Laffel, L., and Christlieb, A. (1991) Am. J. Med. 90, 56S-61S [Medline] [Order article via Infotrieve]
  3. Feldt-Rasmussen, B. (1986) Diabetologia 29, 282-286 [Medline] [Order article via Infotrieve]
  4. Johnson, P. C., Brendel, K., and Meezan, E. (1982) Arch. Pathol. Lab. Med. 60, 214-217
  5. Kuwabara, T., and Kogan, D. G. (1960) Arch. Ophthalmol. 64, 904-911
  6. Yamagishi, S., Kobayashi, K., and Yamamoto, H. (1993) Biochem. Biophys. Res. Commun. 190, 418-425 [CrossRef][Medline] [Order article via Infotrieve]
  7. Shepro, D., and Morel, N. M. (1993) FASEB J. 7, 1031-1038 [Abstract/Free Full Text]
  8. Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F., Pan, Y.-C. E., Elliston, K., Stern, D., and Shaw, A. (1992) J. Biol. Chem. 267, 14998-15004 [Abstract/Free Full Text]
  9. Yamagishi, S., Hsu, C.-C., Taniguchi, M., Harada, S., Yamamoto, Y., Ohsawa, K., Kobayashi, K., and Yamamoto, H. (1995) Biochem. Biophys. Res. Commun. 213, 681-687 [CrossRef][Medline] [Order article via Infotrieve]
  10. Doi, T., Vlassara, H., Kirstein, M., Yamada, Y., Striker, G. E., and Striker, L. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2873-2877 [Abstract]
  11. Esposito, C., Gerlach, H., Brett, J., Stern, D., and Vlassara, H. (1989) J. Exp. Med. 170, 1387-1407 [Abstract]
  12. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  13. Makita, Z., Vlassara, H., Cerami, A., and Bucala, R. (1992) J. Biol. Chem. 267, 5133-5138 [Abstract/Free Full Text]
  14. Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332 [Abstract/Free Full Text]
  15. Micheel, F., and Hagemann, G. (1959) Chem. Ber. 90, 2836-2840
  16. Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894 [Abstract/Free Full Text]
  17. Reddy, S., Bichlar, J., Wells-Knecht, K. J., Thorpe, S. R., and Baynes, J. W. (1995) Biochemistry 34, 10872-10878 [Medline] [Order article via Infotrieve]
  18. Yamagishi, S., Hsu, C.-C., Kobayashi, K., and Yamamoto, H. (1993) Biochem. Biophys. Res. Commun. 191, 840-846 [CrossRef][Medline] [Order article via Infotrieve]
  19. Miyazono, K., Okabe, T., Urabe, A., Takaku, F., and Heldin, C.-H. (1987) J. Biol. Chem. 262, 4098-4103 [Abstract/Free Full Text]
  20. Nomura, M., Yamagishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J., and Yamamoto, H. (1995) J. Biol. Chem. 270, 28316-28324 [Abstract/Free Full Text]
  21. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1412 [Abstract]
  22. Hatakeyama, H., Miyamori, I., Fujita, T., Takeda, Y., Takeda, R., and Yamamoto, H. (1994) J. Biol. Chem. 269, 24316-24320 [Abstract/Free Full Text]
  23. Ooka, H., Chonan, E., Mizutani, K., Fukuda, T., Kuroiwa, Y., Ono, Y., and Shigeta, S. (1992) Microbiol. Immunol. 36, 1305-1316 [Medline] [Order article via Infotrieve]
  24. Kubota, Y., Kleinman, H. K., Martin, G. R., and Lawley, T. J. (1989) J. Cell Biol. 107, 1589-1598 [Abstract]
  25. Grant, D. S., Lelkes, P. I., Fukuda, K., and Kleinman, H. K. (1991) In Vitro Cell. Dev. Biol. 27A, 327-336
  26. Hayase, F., Nagaraj, R. H., Miyata, S., Njoroge, F. G., and Monnier, V. M. (1989) J. Biol. Chem. 264, 3758-3764 [Abstract/Free Full Text]
  27. Gugliucci, A., and Bendayan, M. (1996) Diabetologia 39, 149-160 [Medline] [Order article via Infotrieve]
  28. Nakayama, H., Taneda, S., Kuwajima, S., Aoki, S., Kuroda, Y., Misawa, K., and Nakagawa, S. (1989) Biochem. Biophys. Res. Commun. 162, 740-745 [CrossRef][Medline] [Order article via Infotrieve]
  29. Houck, K. A., Ferrara, N., Winer, J., Cachianes, G., Li, B., and Leung, D. W. (1991) Mol. Endocrinol. 5, 1806-1814 [Abstract]
  30. Ferrara, N., Houck, K. A., Jakeman, L., and Leung, D. W. (1992) Endocr. Rev. 13, 18-32 [Medline] [Order article via Infotrieve]
  31. Termam, B. I., Dougher-Vermazen, M., Carrion, M. E., Dimitrov, D., Armellino, D. C., Gospodarowics, D., and Böhlen, P. (1992) Biochem. Biophys. Res. Commun. 187, 1579-11586 [Medline] [Order article via Infotrieve]
  32. Vries, C. D., Escobedo, J. A., Ueno, H., Houck, K. A., Ferrara, N., and Williams, L. T. (1992) Science 255, 989-991 [Medline] [Order article via Infotrieve]
  33. Folkman, J., and Haudenschild, C. (1980) Nature 288, 551-556 [Medline] [Order article via Infotrieve]
  34. Folkman, J., and Shing, Y. (1992) J. Biol. Chem. 267, 10931-10934 [Free Full Text]
  35. Yamagishi, S., Yamamoto, Y., Harada, S., Hsu, C.-C., and Yamamoto, H. (1996) FEBS Lett. 384, 103-106 [CrossRef][Medline] [Order article via Infotrieve]
  36. Yamamoto, Y., Yamagishi, S., Hsu, C.-C., and Yamamoto, H. (1996) Biochem. Biophys. Res. Commun. 222, 700-705 [CrossRef][Medline] [Order article via Infotrieve]
  37. Namiki, A., Brogi, E., Kearney, M., Kim, E. A., Wu, T., Couffinhal, T., Varticovski, L., and Isner, J. M. (1995) J. Biol. Chem. 270, 31189-31195 [Abstract/Free Full Text]
  38. Simorre-Pinatel, V., Guerrin, M., Chollet, P., Penary, M., Clamens, S., Malecaze, F., and Plouet, J. (1994) Invest. Ophthalmol. & Visual Sci. 35, 3393-3400 [Abstract]
  39. Shima, D. T., Deutsch, U., and D'Amore, P. A. (1995) FEBS Lett. 370, 203-208 [CrossRef][Medline] [Order article via Infotrieve]
  40. Shima, D. T., Adamis, A. P., Ferrara, N., Yeo, K.-T., Yeo, T.-K., Allende, R., Folkman, J., and D'Amore, P. A. (1995) Mol. Med. (Camb.) 1, 182-193 [Medline] [Order article via Infotrieve]
  41. Hammes, H. P., Martin, S., Federlin, K., Geisen, K., and Brownlee, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11555-11558 [Abstract]
  42. Hammes, H. P., Strödter, D., Weiss, A., Bretzel, R. G., Federlin, K., and Brownlee, M. (1995) Diabetologia 38, 656-660 [CrossRef][Medline] [Order article via Infotrieve]
  43. Dolhofer-Bliesener, R., Lechner, B., and Gerbitz, K. D. (1996) Eur. J. Clin. Chem. Clin. Biochem. 34, 355-361 [Medline] [Order article via Infotrieve]
  44. Wautier, J. L., Zoukourian, C., Chappey, O., Wautier, M. P., Guilausseau, P. J., Cao, R., Hori, O., Stern, D., and Schmidt, A. M. (1996) J. Clin. Invest. 97, 238-243 [Abstract/Free Full Text]
  45. Aiello, L. P., Avery, R. L., Arrigg, P. G., Keyt, B. A., Jampel, H. D., Shah, S. T., Pasquale, L. R., Thieme, H., Iwamoto, M. A., Park, J. E., Nguyen, H. V., Aiello, L. M., Ferrara, N., and King, G. L. (1994) N. Engl. J. Med. 331, 1480-1487 [Abstract/Free Full Text]
  46. Adamis, A. P., Miller, J. W., Bernal, M.-T., D'Amico, D. J., Folkman, J., Yeo, T.-K., and Yeo, K.-T. (1994) Am. J. Ophthalmol. 118, 445-450 [Medline] [Order article via Infotrieve]
  47. Dorchy, H. (1993) Diabetes Care 16, 1212-1214 [Medline] [Order article via Infotrieve]
  48. Cho, H. K., Kozu, H., Peyman, G. A., Parry, G. J., and Khoobehi, B. (1990) Ophthalmic Surg. 22, 44-47
  49. Olofsson, B., Pajusola, K., Kaipainen, A., von Euler, G., Joukov, V., Saksela, O., Orpana, A., Pettersson, R. F., Alitalo, K., and Eriksson, U. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2576-2581 [Abstract/Free Full Text]
  50. Joukov, V., Pajusola, K., Kaipainen, A., Chilov, D., Lahtinen, I., Kukk, E., Saksela, O., Kalkkinen, N., and Alitalo, K. (1996) EMBO J. 15, 290-298 [Abstract]
  51. Sato, F., Katsuno, K., Kobayashi, M., Koizumi, T., Baba, Y., Kusama, H., and Iyobe, A. (1996) Diabetes 45, Suppl. 2, 264 (abstr.)

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