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Pigment epithelium-derived factor (PEDF) inhibits collagen-induced platelet activation by reducing intraplatelet nitrotyrosine levels
Letters to the Editor
121
Pigment epithelium-derived factor (PEDF) inhibits collagen-induced platelet activation by reducing intraplatelet nitrotyrosine levels Sho-ichi Yamagishi a,⁎, Takanori Matsui a , Kazuo Nakamura a , Katsuhiko Takenaka b a
Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume, Japan b Department of Medicine, Kurume University School of Medicine, Kurume, Japan Received 6 May 2008; accepted 1 November 2008 Available online 29 November 2008
Abstract We have recently found that pigment epithelium-derived factor (PEDF), a glycoprotein with a potent neuronal differentiating activity, not only inhibits platelet aggregation and P-selectin expression, but also suppresses occlusive thrombus formation in rats through its antioxidative properties. These findings suggest that PEDF may play a protective role against atherothrombosis. However, the underlying molecular mechanism by which PEDF inhibited platelet aggregation and activation in vitro is fully understood. Since nitric oxide (NO) suppresses platelet aggregation and activation, it is conceivable that PEDF could inhibit platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its anti-oxidative properties. In this study, we examined whether PEDF reduced intraplatelet nitrotyrosine levels, a marker of protein nitration by peroxynitrite, and subsequently suppressed platelet-derived growth factorAB (PDGF-AB) production by collage-exposed platelets. PEDF was found to significantly reduce the collagen-elicited intraplatelet nitrotyrosine formation and PDGF-AB secretion by platelets. The present study demonstrated for the first time that PEDF could inhibit the collagen-induced platelet activation by suppressing nitrotyrosine formation. PEDF may inhibit the platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its anti-oxidative properties. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: PEDF; Nitrotyrosine; Platelet activation; PDGF
Pigment epithelium-derived factor (PEDF) is a glycoprotein with potent neuronal differentiating activity [1]. Recently, we have found that PEDF prevents cytokine- or growth factor-elicited endothelial cell damage and smooth muscle cell proliferation and migration [2,3]. In addition, PEDF not only inhibits platelet aggregation and P-selectin expression in vitro, but also suppresses occlusive thrombus formation in rats through its anti-oxidative properties [4]. These observations suggest that PEDF may play a protective role against atherothrombosis. However, the underlying molecular mechanism by which PEDF inhibited platelet aggregation and activation in vitro is fully understood. Platelets possess the L-arginine-nitric oxide (NO) pathway through constitutive NO synthase in humans [5]. There is accumulating evidence to show that NO suppresses platelet aggregation and activation [6]. Since under oxidative stress conditions, NO undergoes a rapid reaction with superoxide anions to form peroxynitrite, a toxic metabolite of NO, which could cause platelet dysfunction [7,8], it is
⁎ 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).
conceivable that PEDF could inhibit platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its anti-oxidative properties. Therefore, in this study, we examined whether PEDF reduced intraplatelet nitrotyrosine levels, a marker of protein nitration by peroxynitrite [7] and subsequently suppressed plateletderived growth factor-AB (PDGF-AB) production by collage-exposed platelets. PEDF proteins were prepared and purified as described previously [9]. 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). Nitrotyrosine expression on platelets was analyzed as described previously [10]. An irrelevant isotype-matched Ab was used as a negative control. Briefly, human platelet-rich plasma (PRP) was pre-incubated with or without 100 nM PEDF. After 60 min, they were stimulated with 2 μg/ml of collagen for 7 min and then fixed with 1% paraformaldehyde. The PRP was incubated with a polyclonal Ab directed against nitrotyrosine as a primary Ab and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G as a second Ab, and then analyzed by flow cytometry. For every histogram, 10,000 platelets were
122
Letters to the Editor
Ministry of Education, Culture, Sports, Science and Technology, Japan (S.Y). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [11]. References
Fig. 1. Effects of PEDF on collagen-induced intraplatelet nitrotyrosine formation (A) and PDGF-AB production by platelets (B). (A) N = 6 per group. (B) N = 4 per group.
counted to evaluate the percentage of positive platelets. For the assay of PDGF-AB, PRP was pre-incubated with or without 100 nM PEDF for 60 min and then with 2 μg/ml collagen. After 7 min, the platelets were precipitated and PDGF-AB content in the supernatant was analyzed by an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA). As shown in Fig. 1A and B, PEDF was found to significantly reduce the collagen-elicited intraplatelet nitrotyrosine formation and PDGF-AB secretion by platelets. The observations suggest that PEDF could inhibit the collageninduced platelet activation by suppressing nitrotyrosine formation. Since we have previously shown that PEDF inhibits the platelet activation and aggregation by suppressing NADPH oxidase-driven reactive oxygen species generation [4], PEDF may inhibit the collagen-evoked platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its antioxidative properties. Acknowledgements This work was supported in part by Grants of Collaboration with Venture Companies Project from the
0167-5273/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2008.11.015
[1] Tombran-Tink J, Barnstable CJ. PEDF: a multifaceted neurotropic factor. Nat Rev Neurosci 2003;4:628–36. [2] Yamagishi S, Nakamura K, Matsui T, Inagaki Y, Takenaka K, Jinnouchi Y, 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. [3] Nakamura K, Yamagishi S, Matsui T, Yoshida T, Takenaka K, Jinnouchi Y, et al. Pigment epithelium-derived factor inhibits neointimal hyperplasia after vascular injury by blocking NADPH oxidase-mediated reactive oxygen species generation. Am J Pathol 2007;170:2159–70. [4] Takenaka K, Yamagishi S, Matsui T, Nakamura K, Jinnouchi Y, Yoshida Y, 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. [5] Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci 1995;57:2049–55. [6] Gkaliagkousi E, Ritter J, Ferro A. Platelet-derived nitric oxide signaling and regulation. Circ Res 2007;101:654–62. [7] Olas B, Wachowicz B. Role of reactive nitrogen species in blood platelet functions. Platelets 2007;18:555–65. [8] Redondo PC, Jardin I, Hernández-Cruz JM, Pariente JA, Salido GM, Rosado JA. Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients. Biochem Biophys Res Commun 2005;333:794–802. [9] 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. [10] Morita H, Ikeda H, Haramaki N, Eguchi H, Imaizumi T. Only twoweek smoking cessation improves platelet aggregability and intraplatelet redox imbalance of long-term smokers. J Am Coll Cardiol 2005;45:589–94. [11] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.
121
Pigment epithelium-derived factor (PEDF) inhibits collagen-induced platelet activation by reducing intraplatelet nitrotyrosine levels Sho-ichi Yamagishi a,⁎, Takanori Matsui a , Kazuo Nakamura a , Katsuhiko Takenaka b a
Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume, Japan b Department of Medicine, Kurume University School of Medicine, Kurume, Japan Received 6 May 2008; accepted 1 November 2008 Available online 29 November 2008
Abstract We have recently found that pigment epithelium-derived factor (PEDF), a glycoprotein with a potent neuronal differentiating activity, not only inhibits platelet aggregation and P-selectin expression, but also suppresses occlusive thrombus formation in rats through its antioxidative properties. These findings suggest that PEDF may play a protective role against atherothrombosis. However, the underlying molecular mechanism by which PEDF inhibited platelet aggregation and activation in vitro is fully understood. Since nitric oxide (NO) suppresses platelet aggregation and activation, it is conceivable that PEDF could inhibit platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its anti-oxidative properties. In this study, we examined whether PEDF reduced intraplatelet nitrotyrosine levels, a marker of protein nitration by peroxynitrite, and subsequently suppressed platelet-derived growth factorAB (PDGF-AB) production by collage-exposed platelets. PEDF was found to significantly reduce the collagen-elicited intraplatelet nitrotyrosine formation and PDGF-AB secretion by platelets. The present study demonstrated for the first time that PEDF could inhibit the collagen-induced platelet activation by suppressing nitrotyrosine formation. PEDF may inhibit the platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its anti-oxidative properties. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: PEDF; Nitrotyrosine; Platelet activation; PDGF
Pigment epithelium-derived factor (PEDF) is a glycoprotein with potent neuronal differentiating activity [1]. Recently, we have found that PEDF prevents cytokine- or growth factor-elicited endothelial cell damage and smooth muscle cell proliferation and migration [2,3]. In addition, PEDF not only inhibits platelet aggregation and P-selectin expression in vitro, but also suppresses occlusive thrombus formation in rats through its anti-oxidative properties [4]. These observations suggest that PEDF may play a protective role against atherothrombosis. However, the underlying molecular mechanism by which PEDF inhibited platelet aggregation and activation in vitro is fully understood. Platelets possess the L-arginine-nitric oxide (NO) pathway through constitutive NO synthase in humans [5]. There is accumulating evidence to show that NO suppresses platelet aggregation and activation [6]. Since under oxidative stress conditions, NO undergoes a rapid reaction with superoxide anions to form peroxynitrite, a toxic metabolite of NO, which could cause platelet dysfunction [7,8], it is
⁎ 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).
conceivable that PEDF could inhibit platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its anti-oxidative properties. Therefore, in this study, we examined whether PEDF reduced intraplatelet nitrotyrosine levels, a marker of protein nitration by peroxynitrite [7] and subsequently suppressed plateletderived growth factor-AB (PDGF-AB) production by collage-exposed platelets. PEDF proteins were prepared and purified as described previously [9]. 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). Nitrotyrosine expression on platelets was analyzed as described previously [10]. An irrelevant isotype-matched Ab was used as a negative control. Briefly, human platelet-rich plasma (PRP) was pre-incubated with or without 100 nM PEDF. After 60 min, they were stimulated with 2 μg/ml of collagen for 7 min and then fixed with 1% paraformaldehyde. The PRP was incubated with a polyclonal Ab directed against nitrotyrosine as a primary Ab and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G as a second Ab, and then analyzed by flow cytometry. For every histogram, 10,000 platelets were
122
Letters to the Editor
Ministry of Education, Culture, Sports, Science and Technology, Japan (S.Y). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [11]. References
Fig. 1. Effects of PEDF on collagen-induced intraplatelet nitrotyrosine formation (A) and PDGF-AB production by platelets (B). (A) N = 6 per group. (B) N = 4 per group.
counted to evaluate the percentage of positive platelets. For the assay of PDGF-AB, PRP was pre-incubated with or without 100 nM PEDF for 60 min and then with 2 μg/ml collagen. After 7 min, the platelets were precipitated and PDGF-AB content in the supernatant was analyzed by an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA). As shown in Fig. 1A and B, PEDF was found to significantly reduce the collagen-elicited intraplatelet nitrotyrosine formation and PDGF-AB secretion by platelets. The observations suggest that PEDF could inhibit the collageninduced platelet activation by suppressing nitrotyrosine formation. Since we have previously shown that PEDF inhibits the platelet activation and aggregation by suppressing NADPH oxidase-driven reactive oxygen species generation [4], PEDF may inhibit the collagen-evoked platelet activation by suppressing the inactivation of NO and subsequent formation of peroxynitrite via its antioxidative properties. Acknowledgements This work was supported in part by Grants of Collaboration with Venture Companies Project from the
0167-5273/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2008.11.015
[1] Tombran-Tink J, Barnstable CJ. PEDF: a multifaceted neurotropic factor. Nat Rev Neurosci 2003;4:628–36. [2] Yamagishi S, Nakamura K, Matsui T, Inagaki Y, Takenaka K, Jinnouchi Y, 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. [3] Nakamura K, Yamagishi S, Matsui T, Yoshida T, Takenaka K, Jinnouchi Y, et al. Pigment epithelium-derived factor inhibits neointimal hyperplasia after vascular injury by blocking NADPH oxidase-mediated reactive oxygen species generation. Am J Pathol 2007;170:2159–70. [4] Takenaka K, Yamagishi S, Matsui T, Nakamura K, Jinnouchi Y, Yoshida Y, 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. [5] Sase K, Michel T. Expression of constitutive endothelial nitric oxide synthase in human blood platelets. Life Sci 1995;57:2049–55. [6] Gkaliagkousi E, Ritter J, Ferro A. Platelet-derived nitric oxide signaling and regulation. Circ Res 2007;101:654–62. [7] Olas B, Wachowicz B. Role of reactive nitrogen species in blood platelet functions. Platelets 2007;18:555–65. [8] Redondo PC, Jardin I, Hernández-Cruz JM, Pariente JA, Salido GM, Rosado JA. Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients. Biochem Biophys Res Commun 2005;333:794–802. [9] 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. [10] Morita H, Ikeda H, Haramaki N, Eguchi H, Imaizumi T. Only twoweek smoking cessation improves platelet aggregability and intraplatelet redox imbalance of long-term smokers. J Am Coll Cardiol 2005;45:589–94. [11] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131: 149–50.