Migration of vascular smooth muscle cells (VSMC) from their normal medial domain into the intimal region of coronary vessels is a vital step in the generation of the atherosclerotic plaque, as well as in coronary artery restenosis following percutaneous coronary angioplasty (PTCA) (Newby & Zaltman, 2000). Elevated plasma concentrations of high-density lipoproteins (HDLs) are associated with a reduced incidence of cardiovascular disease (Boden, 2000), and attenuation of neointimal hyperplasia in experimental animal models (Ameli et al. 1994). We are investigating the role of HDLs on migration of VSMCs in vivo and in vitro. Specifically, we have focused our attention on hepatocyte growth factor (HGF), a known motogen that can regulate the growth and morphogenesis of a variety of cell types and modulate the motility of VSMCs (Morishita et al. 1998). The effects of HDLs on HGF expression are not known. We hypothesised that HDLs can inhibit the expression of HGF, thereby limiting VSMC migration. This may represent one mechanism by which HDLs may protect against neointimal hyperplasia.
To investigate our hypothesis, we examined the effects of HDL pre-incubation on human VSMC secretion of HGF. Human VSMCs, isolated from saphenous vein explants, were subcultured to passages 3-5 in RPMI medium (Sigma, UK) supplemented with 10 % fetal calf serum, glutamine (2 mmol l-1), penicillin (50 U ml-1) and streptomycin (50 µg ml-1), and used at confluence (approximately 2 X 105 cells per well). HDLs (1 mg ml-1 ApoAI) were added to the growth medium of a third of the cultures for an initial 20 h. Tumour necrosis factor-α (TNF-α, 10 ng ml-1) was then added to those cultures and another third of the cultures, and the cells incubated for a further 20 h. The remaining third of the cultures were treated with carrier alone, to give the basal level of production. All media were then harvested for HGF measurement using a sandwich ELISA assay (R&D Systems, UK). The experiment was repeated three times (n = 3 per treatment group per experiment). Statistical significance was evaluated by analysis of variance using Bonferroni correction to adjust for multiple comparisons between the treatment groups. Values are expressed as means ± S.E.M.
In one representative experiment, HGF production was induced approximately threefold in response to TNF-α stimulation, from 0.97 ± 0.048 to 3.32 ± 0.2 ng ml-1 (P < 0.001). Pre-incubation with physiological concentrations of HDLs before the addition of cytokine resulted in a significant reduction in HGF levels to 0.39 ± 0.01 ng ml-1 (P < 0.001). There was no significant difference between the unstimulated control group and those cells treated with HDL before cytokine stimulation (P = 0.13). This is the first demonstration that a cardioprotective lipoprotein can inhibit production of a cytokine-stimulated VSMC motogen. This effect may be important in modulating VSMC migration, an essential step in the pathogenesis of both atherosclerosis and coronary artery restenosis.
Appropiate ethical approval was obtained for use of human saphenous vein explant VSMC cultures. This work was supported by The Wellcome Trust, British Heart Foundation and the Garfield-Weston Foundation.