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A synthetic peptide derived from staphylokinase enhances FGF-2-induced skin wound healing in mice
Thrombosis Research 157 (2017) 7–8
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Letter to the Editors-in-Chief A synthetic peptide derived from staphylokinase enhances FGF-2-induced skin wound healing in mice
Dear Editors,
FGF-2 enhanced Plg activator activities (t-PA and u-PA) determined by fibrin zymography in skin extracts from the injury site of Plg+/+ mice after the injury. However, FGF-2 enhanced u-PA activity in the conditioned media from mouse skin fibroblasts [FGF-2 (1.13 ± 0.07 IU/ml) to control levels (0.031 ± 0.005 IU/ml): 36.5 ± 2.3-fold], although tPA activity in the conditioned media was low with or without FGF-2. From these data, we speculated that FGF-2 enhances skin wound healing mainly through u-PA, but not t-PA, in mice. A three-dimensional invasion assay in the collagen matrix was performed using a double-chamber culture plate to investigate the mechanism by which SP augments skin wound healing accelerated by FGF-2. Mouse skin fibroblasts were seeded in the upper compartment of Transwell chambers with or without SP, FGF-2 (10 ng/ml), and/or Plg (100 μg/ml) for 24 h. FGF-2 treatment enhanced the invasion of mouse fibroblasts. Moreover, SP (10 μg/ml) significantly enhanced FGF-2-induced invasion of fibroblasts in the presence of Plg (Control 83 ± 12 cells, FGF-2251 ± 27 cells*, FGF-2 + SP 328 ± 10 cells**, *p b 0.005, compared with the value of controls, **p b 0.05, compared with the value of FGF-2). These findings suggest that SP enhances the FGF2-induced fibroblastic invasive ability in mouse skin fibroblasts. Extracellular proteases, as well as plasmin and matrix metalloproteases (MMPs), possess pleiotropic functions in tissue repair. Besides the removal of provisional ECM, these proteases exert the liberation of latent growth factor precursors from the cell surface and ECM, the activation of protease-activated receptors, the shedding of growth factor receptors, and the generation of chemotactic degradation products [6]. A previous study indicated that FGF-2 increases the synthesis and secretion of Plg activators from cells, such as fibroblasts [7]. Our and other studies showed that Plg deficiency delayed skin wound healing in mice [8]. Moreover, the present data suggested that FGF-2 enhances u-PA activity in cell extracts and the conditioned media from mouse fibroblasts, as well as fibroblast invasion into the matrix. These findings suggest that FGF-2 increases the u-PA/Plg activation system, inducing ECM degradation and fibroblast invasion in the skin. These events might lead to enhanced skin wound healing in mice. The present study revealed that SP enhances skin wound healing and FGF-2-increased fibroblast invasion into the matrix in Plg+/+ mice. The effects of FGF-2 and/or SP were not observed in Plg−/− mice. These findings suggest that SP enhances FGF-2-induced skin wound healing through a u-PA- and Plg-dependent mechanism. Effective ECM degradation, in the direction of cell migration, requires the co-localization of u-PA and its downstream substrate Plg at the leading
http://dx.doi.org/10.1016/j.thromres.2017.06.032 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
Skin wound healing is divided into four major phases; reaction of hemostasis/inflammation, re-epithelialization/granulation tissue formation, matrix formation, and tissue remodeling [1]. As wound healing progresses, the provisional fibrin matrix by activation of hemostasis is invaded by migrating fibroblasts and keratinocytes degraded by proteinases such as plasmin [2]. The fibrinolytic system involves active serine protease plasmin, and its inactive precursor plasminogen (Plg) is abundantly present in blood plasma, as well as urokinase- and tissue-type plasminogen activators (u-PA and t-PA) [3]. Plasmin, a key factor in the dissolution of fibrin, acts together with other proteases. Plasmin plays a role in the degradation of the extracellular matrix (ECM) in tissue remodeling and cell migration events. On the other hand, streptokinase (SK) and staphylokinase (SAK) from bacteria do not directly convert Plg to plasmin by themselves, but form a stoichiometric complex with plasmin (ogen), and these complexes exhibit Plg activator activity. We previously investigated the interaction between SAK and Plg using peptides synthesized based on the amino acid sequences of SAK [4,5]. In these studies, the nonadecapeptide, GPYLMVNVTGVDGKGNELL, corresponding to Gly22-Leu40 of SAK (abbreviated SP below), bound to Plg and enhanced the activation of Plg by the Plg activator. Moreover, intravenous administration of SP resulted in early recanalization by intrinsic Plg activators in an animal model of carotid artery thrombosis [5]. We therefore investigated the effects of SP derived from SAK with Plg activating ability on wound healing after skin injury by using Plg-deficient (Plg−/−) and wild-type (Plg+/+) mice. Here, we report for the first time the use of this peptide for acceleration of FGF-2-induced skin wound healing in mice. Experimental skin wounding was performed in Plg+/+ and Plg−/− mice. These mice were kindly provided by Professor D Collen (University of Leuven, Leuven, Belgium). Full-thickness wounds (6 mm in diameter) were made on the dorsum of each mouse using a punch biopsy instrument (Maruho, Osaka, Japan). All experiments were performed according to the guidelines of the National Institute of Health and the institutional rules for the use and care of laboratory animals at Kindai University. To observe the natural course of wound healing in both types of mice, the wounds were photographed on days 0 to 14 after the skin injury. Time-dependent alterations of the wound area are shown in Fig. 1A. Re-epithelialization of skin wounds was observed within 14 days in Plg+/+ mice. SP treatment significantly enhanced wound healing increased by FGF-2, although it did not affect wound healing without FGF-2 in Plg+/+ mice. After the effects of FGF-2 and SP on re-epithelialization were determined by hematoxylin and eosin (HE)-stained histological sections at the injured site 11 days after the injury in mice, SP significantly decreased the wound length induced by FGF-2 in Plg+/+ mice (Fig. 1B). On the other hand, wound healing was significantly delayed in Plg−/− mice, compared to those in Plg+/+ mice (Fig. 1A). Moreover, treatment with FGF-2 and/or SP did not affect the wound length in Plg−/− mice (Control 5.6 ± 0.34 mm, FGF-2 5.6 ± 0.41 mm, FGF-2 + SP 5.5 ± 0.23 mm). These findings suggest that SP augments skin wound healing accelerated by FGF-2 through a Plg system in mice.
8
Letter to the Editors-in-Chief
Fig. 1. Skin wound healing in Plg+/+ and Plg−/− mice. (A) The wound area was measured at days 0, 1, 3, 5, 7, 9, 11, and 14 after injury in the presence or absence of local administration of FGF-2 (1 μg) and/or SP (100 μg) in Plg+/+ mice or Plg−/− mice. Each value represents the mean ± SE of 5 mice. *p b 0.05, compared with the value of control (Plg+/+) mice, **p b 0.01, compared with the value of control mice, +p b 0.05, compared with the value of mice treated only with FGF-2. Each value represents the mean ± SE of 5 mice. (B) Upper panel: Representative pictures of hematoxylin- and eosin-stained histological sections at the injured site 11 days after the injury in Plg+/+ mice treated with FGF-2 (1 μg) and/or SP (100 μg). Lower panel: The re-epithelialization length was measured as the distance of the newly formed epidermis that migrated into the wound bed at the injured site 11 days after the injury in Plg+/+ mice. Each value represents the mean ± SE of 5 mice. *p b 0.05, **p b 0.01.
edge of polarized cells [9]. Taken together, SP might bind to Plg on the skin fibroblast surface, inducing activation of the u-PA/Plg system. Our study is the first to show that SP, a synthesis peptide with a Plg activation ability, enhances skin wound healing increased by FGF-2 through the u-PA/Plg system in mice. Further studies are necessary to clarify how the peptide exerts its positive effects on skin wound healing in mice after injury, using mice deficient for several components of the u-PA/Plg system. SP might be clinically useful as a tool to enhance skin wound healing. Conflict of interest statement None. Acknowledgements This work was supported in part by Grants-in Aid for Scientific Research (C) (23590268) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). The authors thank Mrs. Eiko Honda and Mr. Takuya Wada (Life Science Research Institute, Kindai University) for assistance in synthesizing the peptides.
[8] J. Rømer, T.H. Bugge, C. Pyke, L.R. Lund, M.J. Flick, J.L. Degen, Impaired wound healing in mice with a disrupted plasminogen gene, Nat. Med. 2 (1996) 287–292. [9] R.P. Garni-Wagner, B.A. Gyetko, M.R. Todd, H.R. Petty, Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator, Histochem. Cell Biol. 121 (2004) 299–310.
Kiyotaka Okada Division of Basic Medical Science, Kindai University Faculty of Medicine, Osakasayama, Japan Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan Kotarou Kojima Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan Katsumi Okumoto Life Science Research Institute, Kindai University, Osakasayama, Japan Naoyuki Kawao Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan
References [1] A.S. MacLeod, J.N. Mansbridge, The innate immune system in acute and chronic wounds, Adv. Wound Care 4 (2014) 65–78. [2] V. Torisevaa, M. Kahari, Proteinases in cutaneous wound healing, Cell. Mol. Life Sci. 66 (2009) 203–224. [3] D. Collen, H.R. Lijnen, Basic and clinical aspects of fibrinolysis and thrombolysis, Blood 78 (1991) 3114–3124. [4] K. Okada, S. Ueshima, H. Matsuno, N. Kawao, C. Okamoto, M. Tanaka, O. Matsuo, Effect of staphylokinase-derived nonadecapeptide on the activation of plasminogen, Thromb. Haemost. 97 (2007) 795–802. [5] K. Okada, S. Ueshima, H. Matsuno, N. Nagai, N. Kawao, M. Tanaka, O. Matsuo, A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue-type plasminogen activator, J. Thromb. Haemost. 9 (2011) 997–1006. [6] A. Page-McCaw, A.J. Ewald, Z. Werb, Matrix metalloproteinases and the regulation of tissue remodelling, Nat. Rev. Mol. Cell Biol. 8 (2007) 221–233. [7] D. Besser, M. Presta, Y. Nagamine, Elucidation of a signaling pathway induced by FGF2 leading to uPA gene expression in NIH 3T3 fibroblasts, Cell Growth Differ. 6 (1995) 1009–1017.
Osamu Matsuo Kindai University Faculty of Medicine, Osakasayama, Japan Hiroshi Kaji Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan Corresponding author at: Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Ohnohigashi 377-2, Osakasayama City, Osaka 589-8511, Japan. E-mail address: [email protected]. 16 January 2017 Available online 27 June 2017
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Letter to the Editors-in-Chief A synthetic peptide derived from staphylokinase enhances FGF-2-induced skin wound healing in mice
Dear Editors,
FGF-2 enhanced Plg activator activities (t-PA and u-PA) determined by fibrin zymography in skin extracts from the injury site of Plg+/+ mice after the injury. However, FGF-2 enhanced u-PA activity in the conditioned media from mouse skin fibroblasts [FGF-2 (1.13 ± 0.07 IU/ml) to control levels (0.031 ± 0.005 IU/ml): 36.5 ± 2.3-fold], although tPA activity in the conditioned media was low with or without FGF-2. From these data, we speculated that FGF-2 enhances skin wound healing mainly through u-PA, but not t-PA, in mice. A three-dimensional invasion assay in the collagen matrix was performed using a double-chamber culture plate to investigate the mechanism by which SP augments skin wound healing accelerated by FGF-2. Mouse skin fibroblasts were seeded in the upper compartment of Transwell chambers with or without SP, FGF-2 (10 ng/ml), and/or Plg (100 μg/ml) for 24 h. FGF-2 treatment enhanced the invasion of mouse fibroblasts. Moreover, SP (10 μg/ml) significantly enhanced FGF-2-induced invasion of fibroblasts in the presence of Plg (Control 83 ± 12 cells, FGF-2251 ± 27 cells*, FGF-2 + SP 328 ± 10 cells**, *p b 0.005, compared with the value of controls, **p b 0.05, compared with the value of FGF-2). These findings suggest that SP enhances the FGF2-induced fibroblastic invasive ability in mouse skin fibroblasts. Extracellular proteases, as well as plasmin and matrix metalloproteases (MMPs), possess pleiotropic functions in tissue repair. Besides the removal of provisional ECM, these proteases exert the liberation of latent growth factor precursors from the cell surface and ECM, the activation of protease-activated receptors, the shedding of growth factor receptors, and the generation of chemotactic degradation products [6]. A previous study indicated that FGF-2 increases the synthesis and secretion of Plg activators from cells, such as fibroblasts [7]. Our and other studies showed that Plg deficiency delayed skin wound healing in mice [8]. Moreover, the present data suggested that FGF-2 enhances u-PA activity in cell extracts and the conditioned media from mouse fibroblasts, as well as fibroblast invasion into the matrix. These findings suggest that FGF-2 increases the u-PA/Plg activation system, inducing ECM degradation and fibroblast invasion in the skin. These events might lead to enhanced skin wound healing in mice. The present study revealed that SP enhances skin wound healing and FGF-2-increased fibroblast invasion into the matrix in Plg+/+ mice. The effects of FGF-2 and/or SP were not observed in Plg−/− mice. These findings suggest that SP enhances FGF-2-induced skin wound healing through a u-PA- and Plg-dependent mechanism. Effective ECM degradation, in the direction of cell migration, requires the co-localization of u-PA and its downstream substrate Plg at the leading
http://dx.doi.org/10.1016/j.thromres.2017.06.032 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
Skin wound healing is divided into four major phases; reaction of hemostasis/inflammation, re-epithelialization/granulation tissue formation, matrix formation, and tissue remodeling [1]. As wound healing progresses, the provisional fibrin matrix by activation of hemostasis is invaded by migrating fibroblasts and keratinocytes degraded by proteinases such as plasmin [2]. The fibrinolytic system involves active serine protease plasmin, and its inactive precursor plasminogen (Plg) is abundantly present in blood plasma, as well as urokinase- and tissue-type plasminogen activators (u-PA and t-PA) [3]. Plasmin, a key factor in the dissolution of fibrin, acts together with other proteases. Plasmin plays a role in the degradation of the extracellular matrix (ECM) in tissue remodeling and cell migration events. On the other hand, streptokinase (SK) and staphylokinase (SAK) from bacteria do not directly convert Plg to plasmin by themselves, but form a stoichiometric complex with plasmin (ogen), and these complexes exhibit Plg activator activity. We previously investigated the interaction between SAK and Plg using peptides synthesized based on the amino acid sequences of SAK [4,5]. In these studies, the nonadecapeptide, GPYLMVNVTGVDGKGNELL, corresponding to Gly22-Leu40 of SAK (abbreviated SP below), bound to Plg and enhanced the activation of Plg by the Plg activator. Moreover, intravenous administration of SP resulted in early recanalization by intrinsic Plg activators in an animal model of carotid artery thrombosis [5]. We therefore investigated the effects of SP derived from SAK with Plg activating ability on wound healing after skin injury by using Plg-deficient (Plg−/−) and wild-type (Plg+/+) mice. Here, we report for the first time the use of this peptide for acceleration of FGF-2-induced skin wound healing in mice. Experimental skin wounding was performed in Plg+/+ and Plg−/− mice. These mice were kindly provided by Professor D Collen (University of Leuven, Leuven, Belgium). Full-thickness wounds (6 mm in diameter) were made on the dorsum of each mouse using a punch biopsy instrument (Maruho, Osaka, Japan). All experiments were performed according to the guidelines of the National Institute of Health and the institutional rules for the use and care of laboratory animals at Kindai University. To observe the natural course of wound healing in both types of mice, the wounds were photographed on days 0 to 14 after the skin injury. Time-dependent alterations of the wound area are shown in Fig. 1A. Re-epithelialization of skin wounds was observed within 14 days in Plg+/+ mice. SP treatment significantly enhanced wound healing increased by FGF-2, although it did not affect wound healing without FGF-2 in Plg+/+ mice. After the effects of FGF-2 and SP on re-epithelialization were determined by hematoxylin and eosin (HE)-stained histological sections at the injured site 11 days after the injury in mice, SP significantly decreased the wound length induced by FGF-2 in Plg+/+ mice (Fig. 1B). On the other hand, wound healing was significantly delayed in Plg−/− mice, compared to those in Plg+/+ mice (Fig. 1A). Moreover, treatment with FGF-2 and/or SP did not affect the wound length in Plg−/− mice (Control 5.6 ± 0.34 mm, FGF-2 5.6 ± 0.41 mm, FGF-2 + SP 5.5 ± 0.23 mm). These findings suggest that SP augments skin wound healing accelerated by FGF-2 through a Plg system in mice.
8
Letter to the Editors-in-Chief
Fig. 1. Skin wound healing in Plg+/+ and Plg−/− mice. (A) The wound area was measured at days 0, 1, 3, 5, 7, 9, 11, and 14 after injury in the presence or absence of local administration of FGF-2 (1 μg) and/or SP (100 μg) in Plg+/+ mice or Plg−/− mice. Each value represents the mean ± SE of 5 mice. *p b 0.05, compared with the value of control (Plg+/+) mice, **p b 0.01, compared with the value of control mice, +p b 0.05, compared with the value of mice treated only with FGF-2. Each value represents the mean ± SE of 5 mice. (B) Upper panel: Representative pictures of hematoxylin- and eosin-stained histological sections at the injured site 11 days after the injury in Plg+/+ mice treated with FGF-2 (1 μg) and/or SP (100 μg). Lower panel: The re-epithelialization length was measured as the distance of the newly formed epidermis that migrated into the wound bed at the injured site 11 days after the injury in Plg+/+ mice. Each value represents the mean ± SE of 5 mice. *p b 0.05, **p b 0.01.
edge of polarized cells [9]. Taken together, SP might bind to Plg on the skin fibroblast surface, inducing activation of the u-PA/Plg system. Our study is the first to show that SP, a synthesis peptide with a Plg activation ability, enhances skin wound healing increased by FGF-2 through the u-PA/Plg system in mice. Further studies are necessary to clarify how the peptide exerts its positive effects on skin wound healing in mice after injury, using mice deficient for several components of the u-PA/Plg system. SP might be clinically useful as a tool to enhance skin wound healing. Conflict of interest statement None. Acknowledgements This work was supported in part by Grants-in Aid for Scientific Research (C) (23590268) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). The authors thank Mrs. Eiko Honda and Mr. Takuya Wada (Life Science Research Institute, Kindai University) for assistance in synthesizing the peptides.
[8] J. Rømer, T.H. Bugge, C. Pyke, L.R. Lund, M.J. Flick, J.L. Degen, Impaired wound healing in mice with a disrupted plasminogen gene, Nat. Med. 2 (1996) 287–292. [9] R.P. Garni-Wagner, B.A. Gyetko, M.R. Todd, H.R. Petty, Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator, Histochem. Cell Biol. 121 (2004) 299–310.
Kiyotaka Okada Division of Basic Medical Science, Kindai University Faculty of Medicine, Osakasayama, Japan Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan Kotarou Kojima Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan Katsumi Okumoto Life Science Research Institute, Kindai University, Osakasayama, Japan Naoyuki Kawao Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan
References [1] A.S. MacLeod, J.N. Mansbridge, The innate immune system in acute and chronic wounds, Adv. Wound Care 4 (2014) 65–78. [2] V. Torisevaa, M. Kahari, Proteinases in cutaneous wound healing, Cell. Mol. Life Sci. 66 (2009) 203–224. [3] D. Collen, H.R. Lijnen, Basic and clinical aspects of fibrinolysis and thrombolysis, Blood 78 (1991) 3114–3124. [4] K. Okada, S. Ueshima, H. Matsuno, N. Kawao, C. Okamoto, M. Tanaka, O. Matsuo, Effect of staphylokinase-derived nonadecapeptide on the activation of plasminogen, Thromb. Haemost. 97 (2007) 795–802. [5] K. Okada, S. Ueshima, H. Matsuno, N. Nagai, N. Kawao, M. Tanaka, O. Matsuo, A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue-type plasminogen activator, J. Thromb. Haemost. 9 (2011) 997–1006. [6] A. Page-McCaw, A.J. Ewald, Z. Werb, Matrix metalloproteinases and the regulation of tissue remodelling, Nat. Rev. Mol. Cell Biol. 8 (2007) 221–233. [7] D. Besser, M. Presta, Y. Nagamine, Elucidation of a signaling pathway induced by FGF2 leading to uPA gene expression in NIH 3T3 fibroblasts, Cell Growth Differ. 6 (1995) 1009–1017.
Osamu Matsuo Kindai University Faculty of Medicine, Osakasayama, Japan Hiroshi Kaji Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Osakasayama, Japan Corresponding author at: Department of Physiology and Regenerative Medicine, Kindai University Faculty of Medicine, Ohnohigashi 377-2, Osakasayama City, Osaka 589-8511, Japan. E-mail address: [email protected]. 16 January 2017 Available online 27 June 2017