- Email: [email protected]
Pigment epithelium-derived factor (PEDF) inhibits diabetes- or advanced glycation end product (AGE)-induced platelet CD40 ligand overexpression in rats
Letters to the Editor [7] Konings MK, Mali WPTM, Viergever MA. Development of an intravascular impedance catheter for detection of fatty lesions in arteries. IEEE Trans Med Imag 1997;16:439–46. [8] Stiles DK, Oakley B. Simulated characterization of atherosclerotic lesions in the coronary arteries by measurement of bioimpedance. IEEE Trans Biomed Eng 2003;50:916–21. [9] Boone K, Barber D, Brown B. Imaging with electricity: report of the European concerted action on impedance tomography. J Med Eng Technol 1997;21(6):201–32. [10] Jossinet J, Marry E, Matias A. Electrical impedance endotomography. Phys Med Biol 2002;47:2189–202. [11] Plonsey R, Heppner DB. Considerations of quasistationarity in electrophysiological systems. Bull Math Biophys 1967;29(4):657–64. [12] Yang F, Patterson RP. A simulation study on the effect of thoracic conductivity inhomogeneities on sensitivity distributions. Ann Biomed Eng 2008;36(5):762–8. [13] Yang F, Patterson RP. Optimal transvenous coil position on active-can single-coil icd defibrillation efficacy: a simulation study. Ann Biomed Eng 2008;36(1):1659–67.
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[14] Cho S, Thielecke H. Design of electrode array for impedance measurement of lesions in arteries. Physiol Meas 2005;26:S19–26. [15] Calderón AP. On an inverse boundary value problem. Mat Apl Comput 2006;25(2-3):133–8. [16] Lionheart WRB. Conformal uniqueness results in anisotropic electrical impedance imaging. Inverse Problems 1997;13:125–34. [17] Dobson VC, Santosa F. Resolution and stability analysis of an inverse problem in electrical impedance tomography: dependence on the input current patterns. SIAM J Appl Math 1994;54:1542–60. [18] Bioucas-Dias JM, Figueiredo MAT. A new twist: two-step iterative shrinkage/thresholding algorithms for image restoration. IEEE Trans Image Process 2007;16(12):2992–3004. [19] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.
0167-5273/$ - see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2009.01.059
Pigment epithelium-derived factor (PEDF) inhibits diabetes- or advanced glycation end product (AGE)-induced platelet CD40 ligand overexpression in rats Sho-ichi Yamagishi a,⁎, Takanori Matsui a , So Ueda b , Masayoshi Takeuchi c a
c
Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Japan b Department of Medicine, Kurume University School of Medicine, Kurume, Japan Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa, Japan Received 29 December 2008; received in revised form 28 January 2009; accepted 30 January 2009 Available online 23 February 2009
Keywords: PEDF; AGEs; CD40L; Diabetes
Increasing evidence suggests a central role for the CD40– CD40 ligand (CD40L) signaling pathway in the pathogenesis of atherosclerosis [1,2]. Indeed, engagement of CD40 with CD40L evokes inflammatory responses with enhanced expression of adhesion molecules and chemokines in endothelial cells [1,2]. Further, interruption of the CD40– CD40L interactions is reported to inhibit the development and progression of atherosclerosis in animal models [3,4]. ⁎ Corresponding author. Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel./fax: +81 942 31 7873. E-mail address: [email protected] (S. Yamagishi).
CD40L expression is found in activated platelets in vivo, and more than 95% of circulating soluble CD40L originates from platelets [5]. Since elevated CD40L levels are associated with increased risk of cardiovascular events [1,2], it is conceivable that the inhibition of platelet CD40L expression may be a novel therapeutic target for preventing cardiovascular disease (CVD). Altered platelet function and changes in intraplatelet signaling pathways are more prevalent in diabetes [5]. There is a growing body of evidence that advanced glycation end products (AGEs), senescent macroprotein derivatives that form in increased amounts under hyperglycemic conditions, have been implicated in platelet dysfunction, thus
284
Letters to the Editor
contributing to the increased risk for CVD in diabetes [5,6]. We have very recently found that pigment epitheliumderived factor (PEDF), a glycoprotein with potent neuronal cell differentiating activity [7], blocks AGE-elicited endothelial cell damage, vascular inflammation and platelet hyperaggregation through its anti-oxidative properties [8,9]. These observations suggest that PEDF plays a protective role against accelerated atherosclerosis in diabetes by attenuating the deleterious effects of AGEs. However, effects of PEDF on platelet CD40L expression in diabetes remain to be elucidated. Therefore, in this study, we examined the effects of PEDF administration on platelet CD40L expression in diabetic or AGE-injected non-diabetic rats. Further, in order to elucidate the clinical relevance of PEDF administration, we studied whether intraplatelet PEDF levels were actually decreased in diabetic rats. AGE-bovine serum albumin (AGE-BSA) and PEDF proteins were prepared and purified as described previously [9,10]. Briefly, BSA was incubated with 0.1 M 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 sugar. Preparations were tested for endotoxin using Endospecy ES-20S system (Seikagaku Co., Tokyo, Japan); no endotoxin was detectable. SDS-PAGE analysis of purified PEDF proteins revealed a single band with a molecular mass of about 50 kDa, which showed positive reactivity with monoclonal antibody (Ab) directed against human PEDF (Transgenic, Kumamoto, Japan). Streptozotocin-induced diabetic or non-diabetic 9 week-old Sprague–Dawley (SD) rats were injected intravenously with or without 10 µg PEDF every day for up to 7 days as described previously [11]. The rats were killed on the eighth day. Nine week-old SD rats were also injected intravenously with 1 mg AGE-BSA or non-glycated BSA in the presence or absence of 10 µg PEDF every day for up to 10 days as described previously [11]. The rats were killed 1–2 h after injection on the final day. All animal procedures were conducted according to the guidelines provided by the Kurume University Institutional Animal Care and Use Committee under an approved protocol. All values were presented as means± standard error. Unless otherwise indicated, one-way ANOVA followed by the Scheffe F test was performed for statistical comparisons. P b 0.05 was considered significant. CD40L expression on platelets was analyzed as described previously [12]. An irrelevant isotype-matched Ab was used as a negative control. Briefly, human platelet-rich plasma (PRP) was stimulated with 4 µM ADP for 7 min at 37 °C and then fixed with 1% paraformaldehyde. The PRP was incubated with monoclonal Abs directed against CD154 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then analyzed by flow cytometry. For every histogram, 10,000 platelets were counted to evaluate the percentage of positive platelets. Intraplatelet PEDF levels were analyzed with western blots as described previously [12,13]. Briefly, blood was
Fig. 1. Effects of PEDF administration on platelet CD40L expression in diabetic (DM) (A) or non-diabetic AGE-BSA-injected rats (B). Intraplatelet PEDF levels (C). A and B, #P b 0.05, compared to the value with DM or AGE-BSA alone. C, Unpaired t test was performed for statistical comparisons. (A) N = 4 per group, (B) N = 5 per group, (C) N = 6 per group.
collected from rats by venipuncture into a plastic tube containing 3.15% trisodium citrate. Then platelet-rich plasma was prepared and platelets were sedimented according to a previously described method [12]. Platelet proteins were extracted, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane. The membrane was then treated with anti-PEDF antibody. The resultant
Letters to the Editor
immunocomplexes were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and the signal intensities of the bands were measured. Mean blood glucose levels during the experiments in control, diabetic rats and diabetic rats with PEDF were 88.3 ± 3.9, 497.8 ± 49.3 and 540.3 ± 35.2 mg/dl, respectively. There was no significant difference of blood glucose levels in nonglycated BSA-, AGE-BSA- and AGE-BSA plus PEDFinjected rats. Administration of AGEs to normal rats increased its serum levels by about 2-folds; AGE levels in non-glycated BSA-injected vs. AGE-BSA-injected rats were 18.7 ± 0.5 vs. 33.4 ± 1.6 µg/ml, thus indicating that serum AGE concentrations obtained by the AGE injection were comparable to those of diabetic rats [9]. As shown in Fig. 1A, platelet CD40L expression levels were increased by about 2-folds in diabetic rats, which were partly blocked by the treatment with PEDF. Further, AGE injection to non-diabetic rats was found to increase platelet CD40L expression levels by about 1.3-folds, whose effects were completely prevented by the simultaneous treatment with PEDF (Fig. 1B). Although we did not know the exact reasons for the difference of CD40L values between the control (Fig. 1A) and BSA group (Fig. 1B), the difference of treatment conditions (the presence or absence of administration of non-glycated BSA) may account for the discrepancies. Anyway, the present findings suggest that there could exist at least two distinct pathways to platelet CD40L overexpression in diabetes; one is the AGE-dependent pathway which is a molecular target for the anti-platelet effects of PEDF, and the other is the AGE-independent one that could not be suppressed by PEDF treatments. In addition, we found that intraplatelet PEDF levels in diabetic rats were significantly decreased, compared with control rats (Fig. 1C). Taken together, our present study suggests that PEDF could inhibit platelet CD40L overexpression in diabetes by blocking the deleterious effects of AGEs on platelets. Pharmacological up-regulation or substitution of PEDF may offer a novel promising strategy for preventing CVD in diabetes. Further larger scale animal experiment and clinical study are needed to clarify the potential utility of PEDF administration in reducing CVD in diabetes. 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.Y). There is no conflict of interest. 0167-5273/$ - see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2009.01.071
285
The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [14]. References [1] Schonbeck U, Libby P. CD40 signaling and plaque instability. Cir Res 2001;89:1092–103. [2] Vishnervetsky D, Kiyanista VA, Gandhi PJ. CD40 ligand: a novel target in the flight against cardiovascular disease. Ann Pharmacother 2004;38:1500–8. [3] Mach F, Schönbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 1998;394:200–3. [4] Lutgens E, Gorelik L, Daemen MJ, et al. Requirement of CD154 in the progression of atherosclerosis. Mat Med 1999;5:1313–6. [5] Takenaka K, Yamagishi S, Matsui T, Nakamura K, Imaizumi T. Role of advanced glycation end products (AGEs) in thrombogenic abnormalities in diabetes. Curr Neurovasc Res 2006;3:73–7. [6] Yamagishi S, Nakamura K, Matsui T, Ueda S, Fukami K, Okuda S. Agents that block advanced glycation end product (AGE)-RAGE (receptor for AGEs)-oxidative stress system: a novel therapeutic strategy for diabetic vascular complications. Expert Opin Investig Drugs 2008;17:983–96. [7] Tombran-Tink J, Barnstable CJ. PEDF: a multifaceted neurotropic factor. Nat Rev Neurosci 2003;4:628–36. [8] Yamagishi S, Matsui T, Nakamura K, Ueda S, Noda Y, Imaizumi T. Pigment epithelium-derived factor (PEDF): its potential therapeutic implication in diabetic vascular complications. Curr Drug Targets 2008;9: 1025–9. [9] Yamagishi S, Nakamura K, Matsui T, et al. Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression. J Biol Chem 2006;281: 20213–20. [10] Yamagishi S, Inagaki Y, Amano S, Okamoto T, Takeuchi M, Makita Z. Pigment epithelium-derived factor protects cultured retinal pericytes from advanced glycation end products-induced injury through its antioxidative properties. Biochem Biophys Res Commun 2002;296:877–82. [11] Yamagishi S, Matsui T, Nakamura K, Takeuchi M, Imaizumi T. Pigment epithelium-derived factor (PEDF) prevents diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis. Microvasc Res 2006;72:86–90. [12] Takenaka K, Yamagishi S, Matsui T, et al. Pigment epithelium-derived factor (PEDF) administration inhibits occlusive thrombus formation in rats: a possible participation of reduced intraplatelet PEDF in thrombosis of acute coronary syndromes. Atherosclerosis 2008;197:25–33. [13] Yamagishi S, Adachi H, Abe A, et al. Elevated serum levels of pigment epithelium-derived factor in the metabolic syndrome. J Clin Endocrinol Metab 2006;91:2447–50. [14] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.
283
[14] Cho S, Thielecke H. Design of electrode array for impedance measurement of lesions in arteries. Physiol Meas 2005;26:S19–26. [15] Calderón AP. On an inverse boundary value problem. Mat Apl Comput 2006;25(2-3):133–8. [16] Lionheart WRB. Conformal uniqueness results in anisotropic electrical impedance imaging. Inverse Problems 1997;13:125–34. [17] Dobson VC, Santosa F. Resolution and stability analysis of an inverse problem in electrical impedance tomography: dependence on the input current patterns. SIAM J Appl Math 1994;54:1542–60. [18] Bioucas-Dias JM, Figueiredo MAT. A new twist: two-step iterative shrinkage/thresholding algorithms for image restoration. IEEE Trans Image Process 2007;16(12):2992–3004. [19] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.
0167-5273/$ - see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2009.01.059
Pigment epithelium-derived factor (PEDF) inhibits diabetes- or advanced glycation end product (AGE)-induced platelet CD40 ligand overexpression in rats Sho-ichi Yamagishi a,⁎, Takanori Matsui a , So Ueda b , Masayoshi Takeuchi c a
c
Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Japan b Department of Medicine, Kurume University School of Medicine, Kurume, Japan Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa, Japan Received 29 December 2008; received in revised form 28 January 2009; accepted 30 January 2009 Available online 23 February 2009
Keywords: PEDF; AGEs; CD40L; Diabetes
Increasing evidence suggests a central role for the CD40– CD40 ligand (CD40L) signaling pathway in the pathogenesis of atherosclerosis [1,2]. Indeed, engagement of CD40 with CD40L evokes inflammatory responses with enhanced expression of adhesion molecules and chemokines in endothelial cells [1,2]. Further, interruption of the CD40– CD40L interactions is reported to inhibit the development and progression of atherosclerosis in animal models [3,4]. ⁎ Corresponding author. Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel./fax: +81 942 31 7873. E-mail address: [email protected] (S. Yamagishi).
CD40L expression is found in activated platelets in vivo, and more than 95% of circulating soluble CD40L originates from platelets [5]. Since elevated CD40L levels are associated with increased risk of cardiovascular events [1,2], it is conceivable that the inhibition of platelet CD40L expression may be a novel therapeutic target for preventing cardiovascular disease (CVD). Altered platelet function and changes in intraplatelet signaling pathways are more prevalent in diabetes [5]. There is a growing body of evidence that advanced glycation end products (AGEs), senescent macroprotein derivatives that form in increased amounts under hyperglycemic conditions, have been implicated in platelet dysfunction, thus
284
Letters to the Editor
contributing to the increased risk for CVD in diabetes [5,6]. We have very recently found that pigment epitheliumderived factor (PEDF), a glycoprotein with potent neuronal cell differentiating activity [7], blocks AGE-elicited endothelial cell damage, vascular inflammation and platelet hyperaggregation through its anti-oxidative properties [8,9]. These observations suggest that PEDF plays a protective role against accelerated atherosclerosis in diabetes by attenuating the deleterious effects of AGEs. However, effects of PEDF on platelet CD40L expression in diabetes remain to be elucidated. Therefore, in this study, we examined the effects of PEDF administration on platelet CD40L expression in diabetic or AGE-injected non-diabetic rats. Further, in order to elucidate the clinical relevance of PEDF administration, we studied whether intraplatelet PEDF levels were actually decreased in diabetic rats. AGE-bovine serum albumin (AGE-BSA) and PEDF proteins were prepared and purified as described previously [9,10]. Briefly, BSA was incubated with 0.1 M 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 sugar. Preparations were tested for endotoxin using Endospecy ES-20S system (Seikagaku Co., Tokyo, Japan); no endotoxin was detectable. SDS-PAGE analysis of purified PEDF proteins revealed a single band with a molecular mass of about 50 kDa, which showed positive reactivity with monoclonal antibody (Ab) directed against human PEDF (Transgenic, Kumamoto, Japan). Streptozotocin-induced diabetic or non-diabetic 9 week-old Sprague–Dawley (SD) rats were injected intravenously with or without 10 µg PEDF every day for up to 7 days as described previously [11]. The rats were killed on the eighth day. Nine week-old SD rats were also injected intravenously with 1 mg AGE-BSA or non-glycated BSA in the presence or absence of 10 µg PEDF every day for up to 10 days as described previously [11]. The rats were killed 1–2 h after injection on the final day. All animal procedures were conducted according to the guidelines provided by the Kurume University Institutional Animal Care and Use Committee under an approved protocol. All values were presented as means± standard error. Unless otherwise indicated, one-way ANOVA followed by the Scheffe F test was performed for statistical comparisons. P b 0.05 was considered significant. CD40L expression on platelets was analyzed as described previously [12]. An irrelevant isotype-matched Ab was used as a negative control. Briefly, human platelet-rich plasma (PRP) was stimulated with 4 µM ADP for 7 min at 37 °C and then fixed with 1% paraformaldehyde. The PRP was incubated with monoclonal Abs directed against CD154 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then analyzed by flow cytometry. For every histogram, 10,000 platelets were counted to evaluate the percentage of positive platelets. Intraplatelet PEDF levels were analyzed with western blots as described previously [12,13]. Briefly, blood was
Fig. 1. Effects of PEDF administration on platelet CD40L expression in diabetic (DM) (A) or non-diabetic AGE-BSA-injected rats (B). Intraplatelet PEDF levels (C). A and B, #P b 0.05, compared to the value with DM or AGE-BSA alone. C, Unpaired t test was performed for statistical comparisons. (A) N = 4 per group, (B) N = 5 per group, (C) N = 6 per group.
collected from rats by venipuncture into a plastic tube containing 3.15% trisodium citrate. Then platelet-rich plasma was prepared and platelets were sedimented according to a previously described method [12]. Platelet proteins were extracted, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane. The membrane was then treated with anti-PEDF antibody. The resultant
Letters to the Editor
immunocomplexes were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and the signal intensities of the bands were measured. Mean blood glucose levels during the experiments in control, diabetic rats and diabetic rats with PEDF were 88.3 ± 3.9, 497.8 ± 49.3 and 540.3 ± 35.2 mg/dl, respectively. There was no significant difference of blood glucose levels in nonglycated BSA-, AGE-BSA- and AGE-BSA plus PEDFinjected rats. Administration of AGEs to normal rats increased its serum levels by about 2-folds; AGE levels in non-glycated BSA-injected vs. AGE-BSA-injected rats were 18.7 ± 0.5 vs. 33.4 ± 1.6 µg/ml, thus indicating that serum AGE concentrations obtained by the AGE injection were comparable to those of diabetic rats [9]. As shown in Fig. 1A, platelet CD40L expression levels were increased by about 2-folds in diabetic rats, which were partly blocked by the treatment with PEDF. Further, AGE injection to non-diabetic rats was found to increase platelet CD40L expression levels by about 1.3-folds, whose effects were completely prevented by the simultaneous treatment with PEDF (Fig. 1B). Although we did not know the exact reasons for the difference of CD40L values between the control (Fig. 1A) and BSA group (Fig. 1B), the difference of treatment conditions (the presence or absence of administration of non-glycated BSA) may account for the discrepancies. Anyway, the present findings suggest that there could exist at least two distinct pathways to platelet CD40L overexpression in diabetes; one is the AGE-dependent pathway which is a molecular target for the anti-platelet effects of PEDF, and the other is the AGE-independent one that could not be suppressed by PEDF treatments. In addition, we found that intraplatelet PEDF levels in diabetic rats were significantly decreased, compared with control rats (Fig. 1C). Taken together, our present study suggests that PEDF could inhibit platelet CD40L overexpression in diabetes by blocking the deleterious effects of AGEs on platelets. Pharmacological up-regulation or substitution of PEDF may offer a novel promising strategy for preventing CVD in diabetes. Further larger scale animal experiment and clinical study are needed to clarify the potential utility of PEDF administration in reducing CVD in diabetes. 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.Y). There is no conflict of interest. 0167-5273/$ - see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2009.01.071
285
The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [14]. References [1] Schonbeck U, Libby P. CD40 signaling and plaque instability. Cir Res 2001;89:1092–103. [2] Vishnervetsky D, Kiyanista VA, Gandhi PJ. CD40 ligand: a novel target in the flight against cardiovascular disease. Ann Pharmacother 2004;38:1500–8. [3] Mach F, Schönbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature 1998;394:200–3. [4] Lutgens E, Gorelik L, Daemen MJ, et al. Requirement of CD154 in the progression of atherosclerosis. Mat Med 1999;5:1313–6. [5] Takenaka K, Yamagishi S, Matsui T, Nakamura K, Imaizumi T. Role of advanced glycation end products (AGEs) in thrombogenic abnormalities in diabetes. Curr Neurovasc Res 2006;3:73–7. [6] Yamagishi S, Nakamura K, Matsui T, Ueda S, Fukami K, Okuda S. Agents that block advanced glycation end product (AGE)-RAGE (receptor for AGEs)-oxidative stress system: a novel therapeutic strategy for diabetic vascular complications. Expert Opin Investig Drugs 2008;17:983–96. [7] Tombran-Tink J, Barnstable CJ. PEDF: a multifaceted neurotropic factor. Nat Rev Neurosci 2003;4:628–36. [8] Yamagishi S, Matsui T, Nakamura K, Ueda S, Noda Y, Imaizumi T. Pigment epithelium-derived factor (PEDF): its potential therapeutic implication in diabetic vascular complications. Curr Drug Targets 2008;9: 1025–9. [9] Yamagishi S, Nakamura K, Matsui T, et al. Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression. J Biol Chem 2006;281: 20213–20. [10] Yamagishi S, Inagaki Y, Amano S, Okamoto T, Takeuchi M, Makita Z. Pigment epithelium-derived factor protects cultured retinal pericytes from advanced glycation end products-induced injury through its antioxidative properties. Biochem Biophys Res Commun 2002;296:877–82. [11] Yamagishi S, Matsui T, Nakamura K, Takeuchi M, Imaizumi T. Pigment epithelium-derived factor (PEDF) prevents diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis. Microvasc Res 2006;72:86–90. [12] Takenaka K, Yamagishi S, Matsui T, et al. Pigment epithelium-derived factor (PEDF) administration inhibits occlusive thrombus formation in rats: a possible participation of reduced intraplatelet PEDF in thrombosis of acute coronary syndromes. Atherosclerosis 2008;197:25–33. [13] Yamagishi S, Adachi H, Abe A, et al. Elevated serum levels of pigment epithelium-derived factor in the metabolic syndrome. J Clin Endocrinol Metab 2006;91:2447–50. [14] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.