Effect of heparin-derived oligosaccharide on vascular smooth muscle cell proliferation and the signal transduction mechanisms involved.

PURPOSE: In this study, the effect of heparin-derived oligosaccharide (HDO) on vascular endothelial growth factor (VEGF) induced vascular smooth muscle cell (VSMC) proliferation and the signal transduction mechanisms involved were investigated.
METHODS: MTT assays were used to measure VSMC proliferation, flow cytometry to analyze cell cycle distribution, RT-PCR for detection of gene transcript levels, and cell-based ELISA, Western blotting and immunocytochemical methods to detect the expression of PKC-α, ERK 1/2, p-ERK 1/2, Akt, p-Akt, p-PDK1 and p-GSK-3β.
RESULTS: HDO at concentrations of 0.01, 0.1 and 1 μmol·L(-1) dose-dependently inhibited VEGF-induced VSMC proliferation with inhibition indices of 6.8 %, 13.1 % and 28.9 %, respectively. Similar concentrations of HDO dose-dependently decreased the percentage of VEGF-induced cells in S phase to 3.6 %, 3.4 %, and 5.4 %, while increasing that of cells arrested in the G0/G1 phase to 80 %, 82 % and 83.6 %. HDO at 0.01, 0.1 or 1 μmol·L(-1) inhibited VEGF-induced PKC-α mRNA expression, with inhibition indices of 9.2 %, 16.1 % and 54.0 %. HDO at 0.1 or 1 μmol·L(-1) inhibited VEGF-induced proto-oncogene mRNA expression, with inhibition indices of 5.2 % and 6.6 % for c-jun, 8.8 % and 11.6 % for c-myc, and 6.5 % and 11.9 % for c-fos, respectively. Additionally, treatment with 0.01, 0.1 or 1 μmol·L(-1) HDO, inhibited VEGF-induced expression of some proliferation related proteins with inhibition indices of 33.2 %, 56.3 % and 77.0 % for PKC-α, 33.7 %, 38.7 % and 53.2 % for p-Akt, 3.5 %, 24.2 % and 49.3 % for p-ERK 1/2, 39.2 %, 71.8 % and 80.7 % for p-PDK 1 and 41.4 %, 89.4 % and 92.4 % for p-GSK-3β, respectively. The results showed that HDO inhibited PKC-α, c-jun, c-fos and c-myc mRNA transcription, and also down-regulated phosphorylation levels of ERK 1/2 and Akt.
CONCLUSION: Our study demonstrates that HDO inhibits transcription of proliferation-related proto-oncogenes and arrests G1/S transition through inhibition of the PKC, MAPK and Akt/PI3K pathways in association with inhibition of VSMC proliferation. This altered molecular signature may explain one mechanism of HDO-mediated inhibition of VSMC proliferation.