Journal of Korean Endocrine Society 2000;15(2):272-285.
Published online January 1, 2001.
The Effect of Sodium Butyrate (SB), Halofuginone Hydrobromide (HH) and High Glucose Concentration on Cell Growth and Gene Expression in Human Aortic Smooth Muscle Cell.
June Ho Park, Sang Jun Lee, Yeo Jun Ki, Young Jung Jeon, Ki Young Kwon, Hong Suk Song, Seung Beom Han, In Kyu Lee, Jung Chul Kim
1Department of Internal Medicine Keimyung University Dongsan Medical Center, Korea.
2Department of Immunology Kyungpook University School of Medicine1, Taegu, Korea.
Abstract
BACKGROUND
Vascular smooth muscle cell (VSMC) proliferation associated with arterial injury causes restenosis, which remains to be resolved in cardiovascular disease, especially after balloon angioplasty. Although numerous factors including hyperglycemia, hyperinsulinemia, angiotensin, basic fibroblast growth factor (BFGF), etc are suggested as potent mitogens for VSMCs, other mechanisms are still needed to take into new consideration. Advances in molecular biology have led to the development of powerful methods for the analysis of differential gene expression. There, we clarified the effect of glucose, sodium butyrate and halofuginon hydrobromide on gene expression which play a role in VSMC growth. METHODS: Therefore, we evaluate the changes of gene expression in response to high glucose concentration, sodium butyrate which is an inhibitor of platelet-derived growth factor (PDGF), and halofuginon hydrobromide which is an inhibitor of specific type 1 collagen, using differntial expressed sequence tag (EST) sequencing and cDNA microarray hybridization. Human mammary artery VSMC isolated from patients undergoing coronary bypass surgery. Cells from passage 3 to 5 were used in experiment with serum-free media with varying conditions. RESULTS: After 6 days of culture, the cells (VSMC) were resuspended with PBS and counted in a hemocytometer, and viable cells were counted using the trypan blue test. VSMC number reached 36?04 cell under high glucose concentration (H/G: 22mM) and 29?04 cell under low glucose concentration(L/G: 5.5 mM) at 6 day of culture (p<0.01). Sodium butyrate(SB) inhibited VSMC growth at varying butyrate concentrations (0.625, 1.25, 2.5, 5.0, 10.0mM) by 84%, 87%, 94%, 96%, 98%, respectively. Halofuginon hydrobromide(HH) also inhibited VSMC growth at varying halofuginon concentrations (10-11, 10-9, 10-7, 10-5mM) by 15%, 30%, 85%, 100%, respectively. Using a differential EST screening technique to assay the relative level of expression of each of large numbers of cloned cDNA sequences after treatment with high glucose concentration (22mM), sodium butyrate (5 mM), and halofuginon (1microM). Among the total 1,730 cDNA clones, 6 cDNA clones were down-regulated after treatment with sodium butyrate (5mM) and halofuginon (1microM). Those were revealed homology to genes encoding connective tissue growth factor (cTGF), Betaig-H3, nm23-H1 nm23-H2, enigma and copine 1. On the contrary, four clones were up-regulated after treatment with high glucose concentration (22mM). Those clones (BO94-5, K1316-5, K1764-5, B1835-5) didn't match any sequence in the public data base. CONCLUSION: These results indicate that this EST analysis is useful technique in targeting genes which are associated with atherosclerosis in VSMC. These identified clones may be used to assist in the positional cloning of genes which are related with atherosclerosis.
Key Words: Vascular smooth muscle cell, Glucose, Gene expression, EST


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