title: Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration.
abstract: Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
authors.affiliation: Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Korea. jhchung@yonsei.ac.kr
authors.forename: Ji Hyung
authors.initials: JH
authors.lastname: Chung
authors.forename: Eun Kyoung
authors.initials: EK
authors.lastname: Im
authors.forename: Tae Won
authors.initials: TW
authors.lastname: Jin
authors.forename: Seung-Min
authors.initials: SM
authors.lastname: Lee
authors.forename: Soo Hyuk
authors.initials: SH
authors.lastname: Kim
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Choi
authors.forename: Min-Jeong
authors.initials: MJ
authors.lastname: Shin
authors.forename: Kyung Hye
authors.initials: KH
authors.lastname: Lee
authors.forename: Yangsoo
authors.initials: Y
authors.lastname: Jang
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology; D056668:Cathepsin L / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D020552:Comet Assay; D000229:Dependovirus / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases; D007763:Lac Operon / Q000235:genetics; D013058:Mass Spectrometry; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis / Q000235:genetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D057134:Antibodies, Neutralizing; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D048031:JNK Mitogen-Activated Protein Kinases; C534281:CTSL protein, human; D056668:Cathepsin L; D020781:Matrix Metalloproteinase 1