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Connective tissue growth factor induces apoptosis via caspase 3 in cultured human aortic smooth muscle cells

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Abstract

Connective tissue growth factor (CTGF) stimulates proliferation of fibroblasts and endothelial cells, but nothing is known about its role in smooth muscle cells. In this study, the effects of recombinant human CTGF (r-hCTGF, 0.5–10 μg/ml) on cultured human aortic vascular smooth muscle cells were investigated. r-hCTGF significantly reduced cell viability, increased apoptosis, and augmented caspase 3 activity. Moreover, r-hCTGF-induced apoptosis was significantly inhibited by an antibody to CTGF and a caspase-3 inhibitor, Z-Asp(Ome)-Glu-(Ome)Val-Asp(Ome)-FMK. These results suggest that r-hCTGF activates caspase 3 and induces apoptosis.

Introduction

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted peptide (Oemar and Luscher, 1997). CTGF was originally identified in conditioned medium of human umbilical vein endothelial cells (Bradham et al., 1991) and has been shown to play a role in various human diseases including systemic scleroderma (Igarashi et al., 1995), atherosclerosis (Oemar et al., 1997), and renal diseases (Ito et al., 1998). CTGF family members are the immediate early growth-responsive genes that are thought to regulate cell proliferation, differentiation, embryogenesis, and wound healing. Recombinant CTGF protein stimulates type I collagen and fibronectin production in normal rat kidney (NRK) fibroblasts (Frazier et al., 1996). When injected subcutaneously, recombinant CTGF induces granulation and fibrosis in neonatal NIH Swiss mice (Frazier et al., 1996). In addition, the polypeptide stimulates DNA synthesis in cultured fibroblasts (Oemar and Luscher, 1997).

To clarify the effect of CTGF on smooth muscle cells, we treated cultured human aortic smooth muscle cells with recombinant human CTGF (r-hCTGF) and investigated its effect on cell viability and induction of apoptosis.

Section snippets

Cell and materials

Human aortic vascular smooth muscle cells were purchased from KURABO (Tokyo, Japan). Cells were cultured in smooth muscle basal medium (SmBM) (Hishikawa and Luscher, 1998) and the cells in passages 4–8 were used for experiments. All experiments were performed after a 48-h incubation in serum-free Dulbecco's modified eagle medium (DMEM) (GIBCO, Grand Island, NY) with insulin–transferrin–selenite supplement (Hishikawa et al., 1994) (Sigma, St. Louis, MO). r-hCTGF and antibody to CTGF was

Effects of r-hCTGF on cell viability

After a 48-h incubation in serum-free medium, human aortic vascular smooth muscle cells were treated with r-hCTGF for 24–72 h. At 1 μg/ml, r-hCTGF showed no effect until 72 h (Fig. 1). At 5 and 10 μg/ml, r-hCTGF significantly reduced cell viability in a time-dependent manner (Fig. 1A).

To clarify the specificity of the induction of apoptosis by r-hCTGF, antibody to r-hCTGF was used. Pre-treatment with antibody (20 μg/ml) alone had no effect (Fig. 1B). However, the antibody significantly

Discussion

Our results demonstrate the pro-apoptotic effect of r-hCTGF in HASC. CTGF is expressed at very high levels in atherosclerotic but not in normal human blood vessels (Oemar et al., 1997). CTGF expression is localized predominantly in areas with extracellular matrix accumulation, and specially along the shoulder of fibrous caps (Oemar et al., 1997). Interestingly, in human carotid arteries, CTGF-expressing cells are non-proliferating (staining negative for proliferating cell nuclear antigen)

Conclusion

Recombinant human CTGF induces apoptosis in human aortic vascular smooth muscle cells by activating caspase 3. Our results suggest a new therapeutic approach to cardiovascular diseases by modulation of the stability of atherosclerotic lesions with CTGF.

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