Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression

(1)

Angiotensin II augments advanced glycation end

product-induced pericyte apoptosis through RAGE overexpression

Sho-ichi Yamagishi

a,*

, Masayoshi Takeuchi

b

, Takanori Matsui

a

, Kazuo Nakamura

a

,

Tsutomu Imaizumi

a

, Hiroyoshi Inoue

c

a_{Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan} b

Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan

c

Radioisotope Institute for Basic and Clinical Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan Received 1 May 2005; revised 12 June 2005; accepted 24 June 2005

Available online 18 July 2005 Edited by Laszlo Nagy

Abstract

Advanced glycation end product (AGE)-their receptor

(RAGE) and angiotensin II (AII) are implicated in diabetic

ret-inopathy. However, a crosstalk between the two is not fully

understood. In vivo, AGE injection stimulated RAGE expression

in the eye of spontaneously hypertensive rats, which was blocked

by an AII-type 1 receptor blocker, telmisartan. In vitro, AII-type

1 receptor-mediated reactive oxygen species generation elicited

RAGE gene expression in pericytes through NF-

j

B activation.

Further, AII augmented AGE-induced pericyte apoptosis, the

earliest hallmark of diabetic retinopathy. Our present study

may implicate a crosstalk between AGE-RAGE system and

AII in diabetic retinopathy.

Ó

2005 Federation of European Biochemical Societies. Published

Keywords:

Advanced glycation end products; Angiotensin II;

Diabetic retinopathy; Oxidative stress; Pericyte loss; Receptor

for AGEs

1. Introduction

There is a growing body of evidence that hypertension is an

independent risk factor for the incidence and progression of

diabetic retinopathy and that strict blood pressure (BP)

con-trol achieves a clinically important reduction in the risk of

pro-gression of diabetic retinopathy

[1–4]

. Furthermore, recent

clinical studies suggest active participation of the renin–

angiotensin system (RAS) in the pathogenesis of diabetic

retinopathy

[5,6]

. Indeed, interruption of the RAS with

angio-tensin-coverting enzyme inhibitors or angiotensin II (AII)-type

1 receptor blockers (ARBs) has been reported to reduce renal

or retinal disease progression in diabetic patients with

hyper-tension

[7–9]

.

Non-enzymatic modiﬁcation of proteins by reducing sugars,

a process that is also known as Maillard reaction, progress at

an extremely accelerated rate under diabetes, leading to the

formation of advanced glycation end products (AGEs)

in vivo

[10,11]

. Recent understandings of this process have

re-vealed that the AGE-their receptor (RAGE) system plays a

central role in the pathogenesis of diabetic vascular

complica-tion as well

[12–15]

. Engagement of RAGE by AGEs activates

its downstream signaling and subsequently evokes oxidative

stress and inﬂammatory responses in vascular wall cells, thus,

contributing to the development and progression of diabetic

retinopathy

[16–19]

. However, the crosstalk between the

AGE-RAGE system and the RAS in diabetic retinopathy is

not fully understood. In this study, we ﬁrst investigated

whether AII-type 1 receptor interaction was involved in

RAGE expression in the eye of spontaneously hypertensive

rats (SHR). Then we examined whether AII upregulated

RAGE mRNA levels in cultured retinal pericytes in vitro

and the way that it might achieve this eﬀect. We further studied

here whether AII could augment the cytopathic eﬀects of

AGEs on pericytes through RAGE overexpression.

2. Materials and methods

2.1. Materials

AII, diphenylene iodonium (DPI), an inhibitor of NADPH oxidase andN-acetylcysteine (NAC) were purchased from Sigma (St. Louis, MO, USA). Telmisartan, an ARB, was generously gifted from Boeh-ringer Ingelheim (Ingelheim, Germany). GeneAmp RNA PCR Core Kit was from Applied Biosystems (Branchburg, NJ, USA).

2.2. Preparation of AGE-proteins

AGE-bovine serum albumin (BSA) was prepared as described previ-ously[20]. Briefly, BSA was incubated under sterile conditions withD D-glyceraldehyde for 7 days. Then unincorporated sugars were removed by dialysis against phosphate-buffered saline. Control non-glycated BSA was incubated in the same conditions except for the absence of reducing sugars. Preparations were tested for endotoxin using Endosp-ecy ES-20S system (Seikagaku Co., Tokyo, Japan); no endotoxin was detectable. The extent of chemical modification was determined with 2,4,6-trinitrobenzenesulfonic acid and reported as the difference be-tween lysine residues of modified and unmodified protein preparations [20]. The extent of lysine modification (%) of modified BSA prepara-tions was 65% for AGE-BSA.

2.3. Animal study

Seven-week-old normoglycemic SHR (Charles River Breeding Lab-oratories, Yokohama, Japan) were used in this study. After a 1-week adaptation period, rats were given tail vein injections with 2 mg Abbreviations: RAS, renin–angiotensin system; AII, angiotensin II;

ARBs, AII-type 1 receptor blockers; AGEs, advanced glycation end products; RAGE, receptor for AGEs; SHR, spontaneously hyperten-sive rats; DPI, diphenylene iodonium; NAC,N-acetylcysteine; BSA, bovine serum albumin; ROS, reactive oxygen species; RT-PCR, reve-rse transcription-polymerase chain reaction; BP, blood pressure *

Corresponding author.

E-mail address:shoichi@med.kurume-u.ac.jp(S. Yamagishi).

(2)

AGE-BSA (n= 4) or non-glycated BSA (n= 4) or with 2 mg AGE-BSA followed by oral administration of telmisartan (2 mg/kg/day) (n= 4) every day for up to 10 days. Then, the rats were sacrificed 1–2 h after injection on the final day. This AGE administration signif-icantly (P< 0.01) increases serum AGE levels, but telmisartan treat-ment did not affect the AGE levels; the serum AGE levels of non-glycated BSA-treated, AGE-BSA-treated, and AGE-BSA plus tel-misartan-treated SHR were 21.7 ± 0.3, 26.0 ± 0.3, and 25.3 ± 0.3lg/ ml, respectively. All animal procedures were conducted according to the guidelines provided by the Kurume University Institutional Ani-mal Care and Use Committee under an approved protocol.

2.4. Western blotting analysis

Proteins were extracted from enucleated eyes, and then separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellu-lose membranes (Biorad, Hercules, CA, USA) as described previously [21]. Membranes were probed with 1:1000 dilution of anti-serum against RAGE[22]or 1:5000 dilution of monoclonal antibody against a-tubulin (Sigma), and then the immune complexes were visualized with an enhanced chemiluminescence detection system (ECL; Amer-sham Bioscience, Buckinghamshire, United Kingdom).

2.5. Cells

Pericytes were isolated from bovine retina and maintained in Dul-beccoÕs modiﬁed EagleÕs medium containing 5.5 mM glucose (Sigma) supplemented with 20% of fetal bovine serum (ICN Biomedicals Inc., Aurora, OH, USA) as described previously[23]. AGE or AII treatments were carried out in a medium containing 2% fetal bovine serum. 2.6. Measurement of intracellular ROS generation

Pericytes were incubated with or without 100 nM AII in the presence or absence of 100 nM telmisartan or 50 nM DPI for the indicated time periods. Then the intracellular formation of reactive oxygen species (ROS) was measured using the ﬂuorescent probe CM–H2DCFDA (Molecular Probes, Eugene, OR, USA)[24].

2.7. Measurement of luciferase activity

Plasmid, containing the NF-jB promoter attached upstream to the luciferase reporter gene, was kindly provided by Dr. T. Fujita, Depart-ment of Tumor Cell Biology, Tokyo Metropolitan Institute of Medical Science. Luciferase activity was measured as described previously[25]. 2.8. Primers and probes

Sequences of the upstream and downstream primers used in the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were 50_{-ATGGAAACTGAACACAGGCC-3}0 _{and 5}0 -CACACATGTCCCCACCTTAT-30 _{for detecting bovine RAGE} mRNA[26]. Sequences of the primers for humanb-actin mRNAs were the same as described previously[20].

2.9. Semi-quantitative RT-PCR

Poly(A)+RNAs were isolated from pericytes treated with or without 100 nM AII in the presence or absence of 100 nM telmisartan or 1 mM NAC for 4 h, and then analyzed by RT-PCR as described previously [20]. The amounts of poly(A)+_{RNA templates (30 ng) and cycle} num-bers (37 cycles for RAGE gene and 22 cycles forb-actin gene) for ampliﬁcation were chosen in quantitative ranges, where reactions pro-ceeded linearly, which had been determined by plotting signal intensi-ties as functions of the template amounts and cycle numbers[26]. 2.10. Measurement of growth of pericytes

Pericytes were preincubated in the presence or absence of 100 nM AII for 4 h. After washing with phosphate-buﬀered saline once, the cells were treated with various concentrations of AGE-BSA or 100lg/ml non-glycated BSA for 2 days, and then the number of viable cells was determined as described previously[27].

2.11. Measurement of apoptotic cell death in pericytes

Pericytes were preincubated in the presence or absence of 100 nM AII for 4 h. After washing with phosphate-buﬀered saline once, the cells were treated with various concentrations of AGE-BSA or 100lg/ml non-glycated BSA for 2 days. Then the cells were lysed and the

supernatant analyzed in an enzyme-linked immunosorbent assay for DNA fragments (Cell Death Detection enzyme-linked immunosorbent assay, Roche Molecular Biochemicals, Mannheim, Germany) accord-ing to the manufacturerÕs instructions. The amount of apoptosis is shown as the relative absorbance value of the control cells[28]. 2.12. Statistical analysis

All values were presented as means ± S.E. Unless otherwise indi-cated, one-way ANOVA followed by the ScheﬀeFtest was performed. For statistical comparisons of the values between no treatment and AII pretreatment (Fig. 5), unpairedttest was performed;P< 0.05 was con-sidered signiﬁcant.

3. Results

3.1. Eﬀects of telmisartan on RAGE expression in the eye of

SHR

We ﬁrst investigated whether AII-type 1 receptor interaction

was involved in RAGE expression in the eye of SHR. For this,

we examined eﬀects of an ARB, telmisartan on RAGE

expres-sion in the eye of AGE-injected SHR. As shown in

Fig. 1

,

tel-misartan blocked the AGE-induced RAGE expression in the

eye, decreasing its level below that of non-glycated

BSA-in-jected SHR. Telmisartan treatment for 10 days signiﬁcantly

de-creased BP level; systolic BP in non-glycated BSA-treated,

AGE-BSA-treated, and AGE-BSA plus telmisartan-treated

SHR were 194.8 ± 3.7, 195.0 ± 3.6, and 182.8 ± 1.5 mmHg,

respectively. The serum creatinine levels of non-glycated

BSA-treated, AGE-BSA-treated, and AGE-BSA plus

telmisar-tan-treated rats were 0.33 ± 0.02, 0.33 ± 0.02, and 0.35 ±

0.03 mg/dl, respectively.

3.2. Eﬀects of AII on ROS generation in pericytes

We next investigated eﬀects of AII on ROS generation in

cultured pericytes. As shown in

Fig. 2A

, 100 nM AII

signiﬁ-cantly increased ROS generation in pericytes; 2- or 4-h

incuba-tion of 100 nM AII increased intracellular ROS generaincuba-tion by

about 1.5-fold. Telmisartan or DPI, an inhibitor of NADPH

oxidase was found to completely inhibit the AII-induced

in-crease in ROS generation, thus, suggesting that AII-type 1

receptor interaction elicited ROS generation in cultured

peri-cytes via NADPH oxidase activity (

Fig. 1

B). Although high

glucose elicited the mitochondrial ROS generation in pericytes

[29]

, we used here Dulbecco

Õ

s modiﬁed Eagle

Õ

s medium

containing 5.5 mM glucose. Furthermore, there is a growing

Fig. 1. Eﬀects of oral administration of telmisartan on RAGE expression in the eye of SHR. SHR were given tail vein injections with 2 mg AGE-BSA (n= 4) or non-glycated BSA (n= 4) or with 2 mg AGE-BSA followed by oral administration of telmisartan (2 mg/kg/ day) (n= 4) every day for up to 10 days. Then, RAGE expression in the eye was analyzed by Western blots. Similar results were obtained in three independent experiments.

(3)

body of evidence that AII stimulates ROS generation via

NADPH oxidase activity

[30]

. These are reasons why DPI

completely ameliorated ROS production.

3.3. Eﬀects of AII on NF-jB promoter activity in pericytes

ROS modulate a number of gene expressions through the

activation of transcriptional factor NF-

j

B

[31]

. Therefore,

we examined whether AII could induce transcriptional

activa-tion of NF-

j

B in pericytes. As shown in

Fig. 3

, AII

signiﬁ-cantly increased the NF-

j

B promoter activity. Telmisartan

as well as an anti-oxidant NAC was found to completely block

the AII-induced increase in NF-

j

B promoter activity.

Telmi-sartan or NAC alone did not aﬀect NF-

j

B promoter activity

(data not shown). We used here low glucose-containing

Dulbecco

Õ

s modiﬁed Eagle

Õ

s medium. This is a reason why

tel-misartan did not aﬀect the basal NF-

j

B activity in pericytes.

3.4. Eﬀects of AII on RAGE mRNA levels in pericytes

The promoter of RAGE gene was shown to contain diﬀerent

recognition sites for transcription factors NF-

j

B, which

activ-ity was associated with RAGE overexpression in vascular cells

[32,33]

. So, we next studied whether AII could aﬀect gene

expression of RAGE in pericytes. As shown in

Fig. 4

, AII

signiﬁcantly upregulated RAGE mRNA levels, which were

Fig. 2. Eﬀects of AII on ROS generation in pericytes. (A, B) Pericytes were treated with or without 100 nM AII in the presence or absence of 100 nM telmisartan or 50 nM DPI for the indicated time periods, and then ROS were quantitatively analyzed. The percentage of ROS generation is indicated on the ordinate and related to the value for the control with no additives. (A)*_P_{< 0.05 and}**_P_{< 0.01 compared to} the value of the control without AII. (B) *_P_{< 0.05 and} **_P_{< 0.01} compared to the value with AII alone.

Fig. 3. Eﬀects of AII on NF-jB activity in pericytes. Pericytes were treated with or without 100 nM AII in the presence or absence of 100 nM telmisartan or 1 mM NAC for 4 h, and then NF-jB luciferase activity was measured. The percentage of luciferase activity is related to the value of the control.*_P_{< 0.05 and}**_P_{< 0.01 compared to the} value with AII alone.

Fig. 4. Eﬀects of AII on RAGE gene expression in pericytes. Pericytes were treated with or without 100 nM AII in the presence or absence of 100 nM telmisartan or 1 mM NAC for 4 h, and then analyzed by RT-PCR. Lower panels show the quantitative representation of RAGE gene induction. Data were normalized by the intensity of b-actin mRNA-derived signals and related to the value of the control. *_P_{< 0.05 compared to the value with AII alone.}

(4)

completely inhibited by telmisartan or NAC. Telmisartan or

NAC alone did not aﬀect RAGE mRNA levels in pericytes

(data not shown).

3.5. Eﬀects of AII pretreatment on AGE toxicity to pericytes

It is probable that the increased RAGE proteins could

fur-ther transduce the AGE signals, thus, leading to exacerbation

of AGE actions on pericytes. Therefore, we next investigated

eﬀects of AII pretreatment on growth and apoptosis of

peri-cytes. As shown in

Fig. 5A and B

, pretreatment of AII

signif-icantly augmented growth inhibitory as well as pro-apoptotic

eﬀects of AGEs on pericytes.

4. Discussion

In the present study, we demonstrated for the ﬁrst time that

telmisartan, a commercially available ARB, blocked RAGE

overexpression in the eye of AGE-treated SHR. Furthermore,

we found here that AII-type 1 receptor interaction-mediated

ROS generation stimulated RAGE gene induction via

NF-j

B activation, thereby augmenting the cytopathic eﬀects of

AGEs to cultured retinal pericytes. These observations suggest

that there could exist a functional crosstalk between the

AGE-RAGE system and the AII-type 1 receptor in the pathogenesis

of diabetic retinopathy.

In this study, we chose SHR to examine the involvement of

AII-type 1 receptor system in tissue expression of RAGE,

be-cause these rats display an enhanced local RAS, including the

eye

[34–36]

. When SHR were injected intravenously with 2 mg

AGE-BSA for 10 days, which increases serum AGE levels to

those of diabetic situation (unpublished data), RAGE

expres-sion in the eye was increased by about 2-fold (

Fig. 1

).

Telmi-sartan blocked the AGE-induced RAGE overexpression,

thus, decreasing its level below that of non-glycated

BSA-in-jected SHR. These results suggest that both basal and

AGE-elicited RAGE expression in the eye were under the control

of the AII-type 1 receptor system. In the study, telmisartan

treatment did not aﬀect the circulating AGE levels or renal

function. Therefore, it is unlikely that telmisartan treatment

decreases RAGE expression via a reduction in one of its

ligands, AGEs.

In order to elucidate the molecular mechanism for RAGE

upregulation by AII, we did in vitro retinal pericyte

experi-ments, because the above in vivo data cannot completely

ex-clude the possibility that BP lowering eﬀects of telmisartan

could contribute to RAGE suppression in the eye. In this

study, AII signiﬁcantly increased ROS generation in cultured

retinal pericytes, which was blocked by telmisartan or an

inhibitor of NADPH oxidase, DPI. Further, telmisartan or

an anti-oxidant NAC completely prevented the AII-induced

NF-

j

B activation and subsequent upregulation of RAGE

mRNA levels in pericytes. Taken together, these results

sug-gest that blockade by telmisartan of the AII-type 1 receptor

system inhibited RAGE induction in pericytes by suppressing

NADPH oxidase-mediated ROS generation and NF-

j

B

acti-vation. In support of this speculation, redox-sensitive NF-

j

B

promoter activity is reported to be required for the induction

of the RAGE gene expression in vascular cells, including

peri-cytes

[32,33]

. Lander et al. have reported recently that

AGE-RAGE-mediated induction of cellular oxidant stress triggers

a cascade of intracellular signals involving Ras and

mitogen-activated protein kinase, culminating in NF-

j

B activation in

smooth muscle cells, macrovascular counterparts of pericytes

[23,37]

. This pathway could provide a vicious cycle of damage

in diabetic retinopathy.

We also demonstrated here for the ﬁrst time that AII

pre-treatment potentiated the AGE-induced growth retardation

as well as apoptotic cell death of pericytes (

Fig. 5

). Retinal

pericytes play an important role in the maintenance of

micro-vascular homeostasis

[23,38]

. It has been postulated that many

of characteristic changes of diabetic retinopathy are the

conse-quent of the loss of pericytes

[23,24,39]

. Further, the RAS in

the eye is activated in diabetes or hypertension

[40,41]

and that

RAGE mediates the AGE-induced cytotoxicity to retinal

pericytes

[19,26]

. These observations suggest that the RAGE

Fig. 5. Eﬀects of AII pretreatment on viable cell number and apoptosis of AGE-exposed pericytes. Pericytes were preincubated in the presence or absence of 100 nM AII for 4 h. After washing with phosphate-buﬀered saline once, the cells were treated with various concentrations of AGE-BSA or 100lg/ml non-glycated BSA for 2 days. Then the viable cell number (A) and apoptotic cell death (B) of pericytes were measured.+_P_{< 0.05 and}++_P_{< 0.01 compared to the} value of non-treated cells with non-glycated BSA. *_P_{< 0.05 and} **_P_{< 0.01 compared to the value of AII-pretreated cells with} non-glycated BSA.

(5)

induction by AII could play an important role in accelerated

apoptosis of pericytes in diabetic patients with hypertension,

thereby providing a molecular basis for understanding why

the association of diabetes with hypertension increases the risk

of diabetic retinopathy

[1–4]

. Our present study indicates that

blockade by telmisartan of the crosstalk between the

AGE-RAGE system and the RAS may be a promising target for

therapeutic intervention in diabetic retinopathy with

hyperten-sive patients.

Acknowledgment: This work was supported in part by Grants of Collaboration with Venture Companies Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan (S. Yamagishi).

References

[1] Stratton, I.M., Kohner, E.M., Aldington, S.J., Turner, R.C., Holman, R.R., Manley, S.E. and Matthews, D.R. (2001) UKPDS 50: Risk factors for incidence and progression of retinopathy in type II diabetes over 6 years from diagnosis. Diabetologia 44, 156–163.

[2] Matthews, D.R., Stratton, I.M., Aldington, S.J., Holman, R.R. and Kohner, E.M.UK Prospective Diabetes Study Group (2004) Risks of progression of retinopathy and vision loss related to tight blood pressure control in type 2 diabetes mellitus: UKPDS 69. Arch. Ophthalmol. 122, 1631–1640.

[3] UK Prospective Diabetes Study Group (1998) Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes. Br. Med. J. 317, 703–713. [4] Ball, S.G. (2003) Beneﬁts of blood pressure reduction in diabetic

patients. J. Hypertens. 21, S31–S36.

[5] Cooper, M.E. (2004) The role of the renin–angiotensin–aldoste-rone system in diabetes and its vascular complications. Am. J. Hypertens. 17, 16S–20S.

[6] Silverstein, R.L., Fenves, A.Z. and Ram, C.V. (2004) ARBs and target organ protection. Exploring beneﬁts beyond their antihy-pertensive eﬀects. Postgrad. Med. 116, 31–38.

[7] Heart Outcomes Prevention Evaluation Study Investigators (2000) Eﬀects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus: results of the HOPE study and MICRO-HOPE substudy. Lancet 355, 253– 259.

[8] Chaturvedi, N., Sjolie, A.K., Stephenson, J.M., Abrahamian, H., Keipes, M., Castellarin, A., Rogulja-Pepeonik, Z. and Fuller, J.H. (1998) Eﬀect of lisinopril on progression of retinopathy in normotensive people with type 1 diabetes. The EUCLID Study Group. EURODIAB Controlled Trial of Lisinopril in Insulin-Dependent Diabetes Mellitus. Lancet 351, 28–31.

[9] Brenner, B.M., Cooper, M.E., de Zeeuw, D., Keane, W.F., Mitch, W.E., Parving, H.H., Remuzzi, G., Snapinn, S.M., Zhang, Z. and Shahinfar, S.RENAAL Study Investigators (2001) Eﬀects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N. Engl. J. Med. 345, 861– 869.

[10] Dyer, D.G., Blackledge, J.A., Thorpe, S.R. and Baynes, J.W. (1991) Formation of pentosidine during nonenzymatic browning of proteins by glucose. Identiﬁcation of glucose and other carbohydrates as possible precursors of pentosidine in vivo. J. Biol. Chem. 266, 11654–11660.

[11] Grandhee, S.K. and Monnier, V.M. (1991) Mechanism of formation of the Maillard protein cross-link pentosidine. Glucose, fructose, and ascorbate as pentosidine precursors. J. Biol. Chem. 266, 11649–11653.

[12] Schmidt, A.M. and Stern, D. (2000) Atherosclerosis and diabetes: the RAGE connection. Curr. Atheroscler. Rep. 2, 430–436. [13] Stitt, A.W., Bucala, R. and Vlassara, H. (1997) Atherogenesis and

advanced glycation: promotion, progression, and prevention. Ann. NY Acad. Sci. 811, 115–127.

[14] Bierhaus, A., Hofmann, M.A., Ziegler, R. and Nawroth, P.P. (1998) AGEs and their interaction with AGE-receptors in

vascular disease and diabetes mellitus. I. The AGE concept. Cardiovasc. Res. 37, 586–600.

[15] Yamagishi, S., Nakamura, K. and Imaizumi, T. (2005) Advanced glycation end products (AGEs) and diabetic vascular complica-tions. Curr. Diabetes Rev. 1, 93–106.

[16] Wendt, T., Bucciarelli, L., Qu, W., Lu, Y., Yan, S.F., Stern, D.M. and Schmidt, A.M. (2002) Receptor for advanced glycation endproducts (RAGE) and vascular inﬂammation: insights into the pathogenesis of macrovascular complications in diabetes. Curr. Atheroscler. Rep. 4, 228–237.

[17] Yamagishi, S., Takeuchi, M., Inagaki, Y., Nakamura, K. and Imaizumi, T. (2003) Role of advanced glycation end products (AGEs) and their receptor (RAGE) in the pathogenesis of diabetic microangiopathy. Int. J. Clin. Pharm. Res. 23, 129–134. [18] Inagaki, Y., Yamagishi, S., Okamoto, T., Takeuchi, M. and Amano, S. (2003) Pigment epithelium-derived factor prevents advanced glycation end products-induced monocyte chemoat-tractant protein-1 production in microvascular endothelial cells by suppressing intracellular reactive oxygen species generation. Diabetologia 46, 284–287.

[19] Yamagishi, S., Inagaki, Y., Amano, S., Okamoto, T., Takeuchi, M. and Makita, Z. (2002) Pigment epithelium-derived factor protects cultured retinal pericytes from advanced glycation end product-induced injury through its antioxidative properties. Biochem. Biophys. Res. Commun. 296, 877–882.

[20] Yamagishi, S., Inagaki, Y., Okamoto, T., Amano, S., Koga, K., Takeuchi, M. and Makita, Z. (2002) Advanced glycation end product-induced apoptosis and overexpression of vascular endo-thelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells. J. Biol. Chem. 277, 20309–20315. [21] Yamagishi, S., Fujimori, H., Yonekura, H., Yamamoto, Y. and Yamamoto, H. (1998) Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhib-itor-1 in human microvascular endothelial cells. Diabetologia 41, 1435–1441.

[22] Sasaki, N., Takeuchi, M., Chowei, H., Kikuchi, S., Hayashi, Y., Nakano, N., Ikeda, H., Yamagishi, S., Kitamoto, T., Saito, T. and Makita, Z. (2002) Advanced glycation end products (AGE) and their receptor (RAGE) in the brain of patients with Creutzfeldt–Jacob disease with prion plaques. Neurosci. Lett. 326, 117–120.

[23] Yamagishi, S., Kobayashi, K. and Yamamoto, H. (1993) Vascu-lar pericytes not only regulate growth, but also preserve prosta-cyclin-producing ability and protect against lipid peroxide-induced injury of co-cultured endothelial cells. Biochem. Biophys. Res. Commun. 190, 418–425.

[24] Yamagishi, S., Okamoto, T., Amano, S., Inagaki, Y., Koga, K., Koga, M., Choei, H., Sasaki, N., Kikuchi, S., Takeuchi, M. and Makita, Z. (2002) Palmitate-induced apoptosis of microvascular endothelial cells and pericytes. Mol. Med. 8, 178–183.

[25] Okamoto, T., Yamagishi, S., Inagaki, Y., Amano, S., Koga, K., Abe, R., Takeuchi, M., Ohno, S., Yoshimura, A. and Makita, Z. (2002) Angiogenesis induced by advanced glycation end products and its prevention by cerivastatin. FASEB J. 16, 1928–1930. [26] Yamagishi, S., Hsu, C.C., Taniguchi, M., Harada, S., Yamamoto,

Y., Ohsawa, K., Kobayashi, K. and Yamamoto, H. (1995) Receptor-mediated toxicity to pericytes of advanced glycosylation end products: a possible mechanism of pericyte loss in diabetic microangiopathy. Biochem. Biophys. Res. Commun. 213, 681– 687.

[27] Yamagishi, S., Hsu, C.C., Kobayashi, K. and Yamamoto, H. (1993) Endothelin 1 mediates endothelial cell-dependent prolifer-ation of vascular pericytes. Biochem. Biophys. Res. Commun. 191, 840–846.

[28] Yamagishi, S., Amano, S., Inagaki, Y., Okamoto, T., Takeuchi, M. and Makita, Z. (2002) Beraprost sodium, a prostaglandin I2 analogue, protects against advanced gycation end products-induced injury in cultured retinal pericytes. Mol. Med. 8, 546–550. [29] Amano, S., Yamagishi, S., Inagaki, Y., Nakamura, K., Takeuchi, M., Inoue, H. and Imaizumi, T. (2005) Pigment epithelium-derived factor inhibits oxidative stress-induced apoptosis and dysfunction of cultured retinal pericytes. Microvasc. Res. 69, 45– 55.

[30] Griendling, K.K., Minieri, C.A., Ollerenshaw, J.D. and Alexan-der, R.W. (1994) Angiotensin II stimulates NADH and NADPH

(6)

oxidase activity in cultured vascular smooth muscle cells. Cir. Res. 74, 1141–1148.

[31] De Nigris, F., Lerman, L.O., Condorelli, M., Lerman, A. and Napoli, C. (2001) Oxidation-sensitive transcription factors and molecular mechanisms in the arterial wall. Antioxidant Redox Signal. 6, 1119–1130.

[32] Tanaka, N., Yonekura, H., Yamagishi, S., Fujimori, H., Yamam-oto, Y. and YamamYamam-oto, H. (2001) The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-alpha through nuclear factor-kappa B, and by 17beta-estradiol through Sp-1 in human vascular endothelial cells. J. Biol. Chem. 275, 25781–25790. [33] Li, J. and Schmidt, A.M. (1997) Characterization and functional

analysis of the promoter of RAGE, the receptor for advanced glycation end products. J. Biol. Chem. 272, 16498–16506. [34] Brown, L., Passmore, M., Duce, B. and Sernia, C. (1997)

Angiotensin receptors in cardiac and rena hypertrophy in rats. J. Mol. Cell Cardiol. 29, 2925–2929.

[35] Jandeleit-Dahm, K., Burrell, L.M., Johnston, C.I. and Koch, K.M. (1997) Elevated vascular angiotensin converting enzyme mediates increased neointima formation after balloon injury in spontaneously hypertensive rats. J. Hypertens. 15, 643–650.

[36] Gin, T., Joon, T.L., Panagiotopoulos, S., Cooper, M., Taylor, H. and Jerums, G. (1996) Organ speciﬁcity of antihypertensive therapy on ocular albumin vascular clearance and albuminuria in the hypertensive diabetic rats. Invest. Ophthalmol. Vis. Sci. 37, 281–289.

[37] Lander, H.M., Tauras, J.M., Ogiste, J.S., Hori, O., Moss, R.A. and Schmidt, A.M. (1997) Activation of the receptor for advanced glycation end products triggers a p21(ras)-dependent mitogen-activated protein kinase pathway regulated by oxidant stress. J. Biol. Chem. 272, 17810–17814.

[38] Antonelli-Orlidge, A., Saunders, K.B., Smith, S.R. and DÕAmore, P.A. (1989) An active form of transforming growth factor-bwas produced by co-cultures of ECs and pericytes. Proc. Natl. Acad. Sci. USA 86, 4544–4548.

[39] Cogan, D.G., Toussaint, D. and Kuwabara, T. (1961) Retinal vascular patterns. IV. Diabetic retinopathy. Arch. Ophthalmol. 66, 366–378.

[40] Anderson, S. (1997) Role of local and systemic angiotensin in diabetic renal disease. Kidney Int. Suppl. 63, S107–S110. [41] Sjolie, A.K. and Chaturvedi, N. (2002) The retinal renin–

angiotensin system: implications for therapy in diabetic retinop-athy. J. Hum. Hypertens. 16 (Suppl 3), S42–S46.