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

Ji Hyung Chung,Eun Kyoung Im,Min-Jeong Shin,Yangsoo Jang,Seung-Min Lee,Soo Hyuk Kim,Kyung Hye Lee,Taewon Jin,Eun Young Choi

doi:10.3858/emm.2011.43.4.022

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.

Highlights

Angiogenesis is the biological process of growing new blood vessels from preexisting vessels, which involves the disruption of the extracellular matrix (ECM) by proteolytic enzymes, endothelial cell proliferation and migration, and the subsequent formation and maturation of new capillary tubes (Markkanen et al, 2005; Chen et al, 2009)
BFGF has a wide spectrum of target cells, including smooth muscle cells, fibroblasts, keratinocytes, and skeletal muscle cells (SkMCs) involved in diverse biological responses (Barrientos et al, 2008), suggesting that endothelial cell migration is probably regulated by communication between the endothelium and the surrounding environment
The results of the present study provide important evidence in support of roles for factors released from SkMCs transfected with the basic fibroblast growth factor (bFGF) gene in the regulation of endothelial cell migration

Summary

Introduction

Angiogenesis is the biological process of growing new blood vessels from preexisting vessels, which involves the disruption of the extracellular matrix (ECM) by proteolytic enzymes, endothelial cell proliferation and migration, and the subsequent formation and maturation of new capillary tubes (Markkanen et al, 2005; Chen et al, 2009). Endothelial cell migration is essential to angiogenesis, which is regulated by chemotactic, haptotactic, and mechanotactic stimuli (Lamalice et al, 2007). Several growth factors contribute to proliferative and migratory events, bFGF plays a key role in endothelial cell migration through its cognate receptor and intracellular signaling response (Pintucci et al, 2002). BFGF has a wide spectrum of target cells, including smooth muscle cells, fibroblasts, keratinocytes, and SkMCs involved in diverse biological responses (Barrientos et al, 2008), suggesting that endothelial cell migration is probably regulated by communication between the endothelium and the surrounding environment

Methods

Results

Conclusion