Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

• Published in: Experimental & molecular medicine 2011, 43(4), , pp.179-188
• Main Authors: Chung, Ji Hyung, Im, Eun Kyoung, Jin, Taewon, Lee, Seung-Min, Kim, Soo Hyuk, Choi, Eun Young, Shin, Min-Jeong, Lee, Kyung Hye, Jang, Yangsoo
• Format: Article
• Language: English
• Subjects: Adenovirus; Antibodies, Neutralizing - immunology; Biomedical and Life Sciences; Biomedicine; Cathepsin L - genetics; Cathepsin L - metabolism; Cell Movement; Cells, Cultured; Comet Assay; Dependovirus - genetics; Endothelial Cells - cytology; Endothelial Cells - metabolism; Fibroblast Growth Factor 2 - genetics; Fibroblast Growth Factor 2 - immunology; Fibroblast Growth Factor 2 - metabolism; Gene Transfer Techniques; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Lac Operon - genetics; Mass Spectrometry; Matrix Metalloproteinase 1 - biosynthesis; Matrix Metalloproteinase 1 - genetics; Medical Biochemistry; Molecular Medicine; Muscle, Skeletal - metabolism; Neovascularization, Physiologic; Original; Plasminogen Activator Inhibitor 1 - biosynthesis; Plasminogen Activator Inhibitor 1 - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; Stem Cells; 생화학
• ISSN: 1226-3613; 2092-6413
• DOI: 10.3858/emm.2011.43.4.022

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.