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Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression
FEBS 29765
FEBS Letters 579 (2005) 4265–4270
Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression Sho-ichi Yamagishia,*, Masayoshi Takeuchib, Takanori Matsuia, Kazuo Nakamuraa, Tsutomu Imaizumia, Hiroyoshi Inouec a
c
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 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 retinopathy. 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-jB 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 by Elsevier B.V. All rights reserved. Keywords: Advanced glycation end products; Angiotensin II; Diabetic retinopathy; Oxidative stress; Pericyte loss; Receptor for AGEs
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 revealed that the AGE-their receptor (RAGE) system plays a central role in the pathogenesis of diabetic vascular complication 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.
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) control achieves a clinically important reduction in the risk of progression 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 angiotensin-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 hypertension [7–9]. Non-enzymatic modification of proteins by reducing sugars, a process that is also known as Maillard reaction, progress at * Corresponding author. E-mail address: [email protected] (S. Yamagishi).
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 hypertensive rats; DPI, diphenylene iodonium; NAC, N-acetylcysteine; BSA, bovine serum albumin; ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction; BP, blood pressure
2. Materials and methods 2.1. Materials AII, diphenylene iodonium (DPI), an inhibitor of NADPH oxidase and N-acetylcysteine (NAC) were purchased from Sigma (St. Louis, MO, USA). Telmisartan, an ARB, was generously gifted from Boehringer 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 previously [20]. Briefly, BSA was incubated under sterile conditions with 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 Endospecy 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 between lysine residues of modified and unmodified protein preparations [20]. The extent of lysine modification (%) of modified BSA preparations was 65% for AGE-BSA. 2.3. Animal study Seven-week-old normoglycemic SHR (Charles River Breeding Laboratories, Yokohama, Japan) were used in this study. After a 1-week adaptation period, rats were given tail vein injections with 2 mg
0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.06.058
4266 AGE-BSA (n = 4) or non-glycated BSA (n = 4) or with 2 mg AGEBSA 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 significantly (P < 0.01) increases serum AGE levels, but telmisartan treatment did not affect the AGE levels; the serum AGE levels of non-glycated BSA-treated, AGE-BSA-treated, and AGE-BSA plus telmisartan-treated SHR were 21.7 ± 0.3, 26.0 ± 0.3, and 25.3 ± 0.3 lg/ ml, respectively. All animal procedures were conducted according to the guidelines provided by the Kurume University Institutional Animal 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 nitrocellulose 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; Amersham Bioscience, Buckinghamshire, United Kingdom). 2.5. Cells Pericytes were isolated from bovine retina and maintained in DulbeccoÕs 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, Department 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 5 0 -ATGGAAACTGAACACAGGCC-3 0 and 5 0 CACACATGTCCCCACCTTAT-3 0 for detecting bovine RAGE mRNA [26]. Sequences of the primers for human b-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 numbers (37 cycles for RAGE gene and 22 cycles for b-actin gene) 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 [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 100 lg/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 100 lg/ml non-glycated BSA for 2 days. Then the cells were lysed and the
S. Yamagishi et al. / FEBS Letters 579 (2005) 4265–4270 supernatant analyzed in an enzyme-linked immunosorbent assay for DNA fragments (Cell Death Detection enzyme-linked immunosorbent assay, Roche Molecular Biochemicals, Mannheim, Germany) according 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 indicated, one-way ANOVA followed by the Scheffe F test was performed. For statistical comparisons of the values between no treatment and AII pretreatment (Fig. 5), unpaired t test was performed; P < 0.05 was considered 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 expression in the eye of AGE-injected SHR. As shown in Fig. 1, telmisartan blocked the AGE-induced RAGE expression in the eye, decreasing its level below that of non-glycated BSA-injected SHR. Telmisartan treatment for 10 days significantly decreased 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 telmisartan-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 significantly increased ROS generation in pericytes; 2- or 4-h incubation of 100 nM AII increased intracellular ROS generation by about 1.5-fold. Telmisartan or DPI, an inhibitor of NADPH oxidase was found to completely inhibit the AII-induced increase in ROS generation, thus, suggesting that AII-type 1 receptor interaction elicited ROS generation in cultured pericytes via NADPH oxidase activity (Fig. 1B). Although high glucose elicited the mitochondrial ROS generation in pericytes [29], we used here DulbeccoÕs modified EagleÕs 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.
S. Yamagishi et al. / FEBS Letters 579 (2005) 4265–4270
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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.
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-jB, which activity 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.
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-jB [31]. Therefore, we examined whether AII could induce transcriptional activation of NF-jB in pericytes. As shown in Fig. 3, AII significantly increased the NF-jB promoter activity. Telmisartan as well as an anti-oxidant NAC was found to completely block the AII-induced increase in NF-jB promoter activity. Telmisartan or NAC alone did not affect NF-jB promoter activity (data not shown). We used here low glucose-containing DulbeccoÕs modified EagleÕs medium. This is a reason why telmisartan did not affect the basal NF-jB activity in pericytes.
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 RTPCR. 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.
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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 further 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 pericytes. As shown in Fig. 5A and B, pretreatment of AII significantly augmented growth inhibitory as well as pro-apoptotic effects of AGEs on pericytes.
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 100 lg/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 nonglycated BSA.
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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 NFjB activation, thereby augmenting the cytopathic effects of AGEs to cultured retinal pericytes. These observations suggest that there could exist a functional crosstalk between the AGERAGE 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, because 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 expression in the eye was increased by about 2-fold (Fig. 1). Telmisartan blocked the AGE-induced RAGE overexpression, thus, decreasing its level below that of non-glycated BSA-injected SHR. These results suggest that both basal and AGEelicited 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 experiments, because the above in vivo data cannot completely exclude 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-jB activation and subsequent upregulation of RAGE mRNA levels in pericytes. Taken together, these results suggest 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-jB activation. In support of this speculation, redox-sensitive NF-jB promoter activity is reported to be required for the induction of the RAGE gene expression in vascular cells, including pericytes [32,33]. Lander et al. have reported recently that AGERAGE-mediated induction of cellular oxidant stress triggers a cascade of intracellular signals involving Ras and mitogenactivated protein kinase, culminating in NF-jB 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 pretreatment 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 microvascular homeostasis [23,38]. It has been postulated that many of characteristic changes of diabetic retinopathy are the consequent 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
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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 AGERAGE system and the RAS may be a promising target for therapeutic intervention in diabetic retinopathy with hypertensive 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).
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FEBS Letters 579 (2005) 4265–4270
Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression Sho-ichi Yamagishia,*, Masayoshi Takeuchib, Takanori Matsuia, Kazuo Nakamuraa, Tsutomu Imaizumia, Hiroyoshi Inouec a
c
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 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 retinopathy. 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-jB 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 by Elsevier B.V. All rights reserved. Keywords: Advanced glycation end products; Angiotensin II; Diabetic retinopathy; Oxidative stress; Pericyte loss; Receptor for AGEs
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 revealed that the AGE-their receptor (RAGE) system plays a central role in the pathogenesis of diabetic vascular complication 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.
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) control achieves a clinically important reduction in the risk of progression 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 angiotensin-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 hypertension [7–9]. Non-enzymatic modification of proteins by reducing sugars, a process that is also known as Maillard reaction, progress at * Corresponding author. E-mail address: [email protected] (S. Yamagishi).
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 hypertensive rats; DPI, diphenylene iodonium; NAC, N-acetylcysteine; BSA, bovine serum albumin; ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction; BP, blood pressure
2. Materials and methods 2.1. Materials AII, diphenylene iodonium (DPI), an inhibitor of NADPH oxidase and N-acetylcysteine (NAC) were purchased from Sigma (St. Louis, MO, USA). Telmisartan, an ARB, was generously gifted from Boehringer 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 previously [20]. Briefly, BSA was incubated under sterile conditions with 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 Endospecy 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 between lysine residues of modified and unmodified protein preparations [20]. The extent of lysine modification (%) of modified BSA preparations was 65% for AGE-BSA. 2.3. Animal study Seven-week-old normoglycemic SHR (Charles River Breeding Laboratories, Yokohama, Japan) were used in this study. After a 1-week adaptation period, rats were given tail vein injections with 2 mg
0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.06.058
4266 AGE-BSA (n = 4) or non-glycated BSA (n = 4) or with 2 mg AGEBSA 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 significantly (P < 0.01) increases serum AGE levels, but telmisartan treatment did not affect the AGE levels; the serum AGE levels of non-glycated BSA-treated, AGE-BSA-treated, and AGE-BSA plus telmisartan-treated SHR were 21.7 ± 0.3, 26.0 ± 0.3, and 25.3 ± 0.3 lg/ ml, respectively. All animal procedures were conducted according to the guidelines provided by the Kurume University Institutional Animal 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 nitrocellulose 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; Amersham Bioscience, Buckinghamshire, United Kingdom). 2.5. Cells Pericytes were isolated from bovine retina and maintained in DulbeccoÕs 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, Department 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 5 0 -ATGGAAACTGAACACAGGCC-3 0 and 5 0 CACACATGTCCCCACCTTAT-3 0 for detecting bovine RAGE mRNA [26]. Sequences of the primers for human b-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 numbers (37 cycles for RAGE gene and 22 cycles for b-actin gene) 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 [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 100 lg/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 100 lg/ml non-glycated BSA for 2 days. Then the cells were lysed and the
S. Yamagishi et al. / FEBS Letters 579 (2005) 4265–4270 supernatant analyzed in an enzyme-linked immunosorbent assay for DNA fragments (Cell Death Detection enzyme-linked immunosorbent assay, Roche Molecular Biochemicals, Mannheim, Germany) according 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 indicated, one-way ANOVA followed by the Scheffe F test was performed. For statistical comparisons of the values between no treatment and AII pretreatment (Fig. 5), unpaired t test was performed; P < 0.05 was considered 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 expression in the eye of AGE-injected SHR. As shown in Fig. 1, telmisartan blocked the AGE-induced RAGE expression in the eye, decreasing its level below that of non-glycated BSA-injected SHR. Telmisartan treatment for 10 days significantly decreased 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 telmisartan-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 significantly increased ROS generation in pericytes; 2- or 4-h incubation of 100 nM AII increased intracellular ROS generation by about 1.5-fold. Telmisartan or DPI, an inhibitor of NADPH oxidase was found to completely inhibit the AII-induced increase in ROS generation, thus, suggesting that AII-type 1 receptor interaction elicited ROS generation in cultured pericytes via NADPH oxidase activity (Fig. 1B). Although high glucose elicited the mitochondrial ROS generation in pericytes [29], we used here DulbeccoÕs modified EagleÕs 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.
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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.
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-jB, which activity 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.
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-jB [31]. Therefore, we examined whether AII could induce transcriptional activation of NF-jB in pericytes. As shown in Fig. 3, AII significantly increased the NF-jB promoter activity. Telmisartan as well as an anti-oxidant NAC was found to completely block the AII-induced increase in NF-jB promoter activity. Telmisartan or NAC alone did not affect NF-jB promoter activity (data not shown). We used here low glucose-containing DulbeccoÕs modified EagleÕs medium. This is a reason why telmisartan did not affect the basal NF-jB activity in pericytes.
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 RTPCR. 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.
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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 further 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 pericytes. As shown in Fig. 5A and B, pretreatment of AII significantly augmented growth inhibitory as well as pro-apoptotic effects of AGEs on pericytes.
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 100 lg/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 nonglycated BSA.
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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 NFjB activation, thereby augmenting the cytopathic effects of AGEs to cultured retinal pericytes. These observations suggest that there could exist a functional crosstalk between the AGERAGE 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, because 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 expression in the eye was increased by about 2-fold (Fig. 1). Telmisartan blocked the AGE-induced RAGE overexpression, thus, decreasing its level below that of non-glycated BSA-injected SHR. These results suggest that both basal and AGEelicited 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 experiments, because the above in vivo data cannot completely exclude 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-jB activation and subsequent upregulation of RAGE mRNA levels in pericytes. Taken together, these results suggest 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-jB activation. In support of this speculation, redox-sensitive NF-jB promoter activity is reported to be required for the induction of the RAGE gene expression in vascular cells, including pericytes [32,33]. Lander et al. have reported recently that AGERAGE-mediated induction of cellular oxidant stress triggers a cascade of intracellular signals involving Ras and mitogenactivated protein kinase, culminating in NF-jB 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 pretreatment 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 microvascular homeostasis [23,38]. It has been postulated that many of characteristic changes of diabetic retinopathy are the consequent 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
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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 AGERAGE system and the RAS may be a promising target for therapeutic intervention in diabetic retinopathy with hypertensive 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).
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