J Korean Soc Endocrinol.  2000 Jun;15(2):272-285.

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

Affiliations
  • 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.


MeSH Terms

Angioplasty, Balloon
Angiotensins
Atherosclerosis
Butyrates
Butyric Acid*
Cardiovascular Diseases
Clone Cells
Cloning, Organism
Collagen Type I
Connective Tissue Growth Factor
Culture Media, Serum-Free
DNA, Complementary
Expressed Sequence Tags
Fibroblast Growth Factor 2
Gene Expression*
Glucose*
Humans*
Hyperglycemia
Hyperinsulinism
Mammary Arteries
Mass Screening
Mitogens
Molecular Biology
Muscle, Smooth*
Muscle, Smooth, Vascular
Myocytes, Smooth Muscle*
Oligonucleotide Array Sequence Analysis
Platelet-Derived Growth Factor
Sodium*
Trypan Blue
Angiotensins
Butyrates
Butyric Acid
Collagen Type I
Connective Tissue Growth Factor
Culture Media, Serum-Free
DNA, Complementary
Fibroblast Growth Factor 2
Glucose
Mitogens
Platelet-Derived Growth Factor
Sodium
Trypan Blue
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