J Korean Soc Endocrinol.  2006 Aug;21(4):302-310. 10.3803/jkes.2006.21.4.302.

The Effect of Epicatechin on the High Glucose-induced TSP-1 Expression and MMP-2 Activity in Rat Vascular Smooth Muscle Cells

• Ryu GR
• Kang JH
• Hong KJ
• Jeong IK
• Jang HI
• Rhie DJ
• Yoon SH
• Hahn SJ
• Kim MS
• Jo YH
• Kim MJ

• KMID: 2200845
• DOI: http://doi.org/10.3803/jkes.2006.21.4.302

Abstract

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs.
METHODS
VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs.
RESULTS
Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion.
CONCLUSION
Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.

MeSH Terms

Animals
Antioxidants
Aorta
Atherosclerosis
Blood Glucose
Blotting, Northern
Blotting, Western
Catechin*
Diabetes Mellitus, Type 2
Gelatin
Glucose
Humans
Incidence
Matrix Metalloproteinase 2
Muscle, Smooth, Vascular*
Rats*
Rats, Sprague-Dawley
RNA, Messenger
Thrombospondin 1*
Antioxidants
Catechin
Gelatin
Glucose
Matrix Metalloproteinase 2
RNA, Messenger
Thrombospondin 1

Figure

• 

  Fig. 1 The effects of high glucose on the expression of thrombospondin-1 (TSP-1) protein in rat aortic vascular smooth muscle cells (VSMCs). VSMCs were incubated with glucose (GLU, 5.6, 11.1, and 30 mM) for 48 h. (A) Total proteins (30 µg) of cellular lysate (cytosol) and medium were separated and analyzed on a 8% SDS-PAGE, and Western blot analysis was performed as described in Methods. Equal loading of cellular lysate was verified by probing the same blot for β-tubulin. (B) TSP-1 levels of cytosol and medium were determined by scanning densitometry of immunoblots. TSP-1 levels of cytosol and media under 5.6 mM glucose were set to 1. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 vs. 5.6 mM GLU.

• 

  Fig. 2 The effects of epicatechin (EC) on high glucoseinduced thrombospondin-1 (TSP-1) protein in rat aortic vascular smooth muscle cells (VSMCs). VSMCs were incubated with glucose (GLU, 5.6 and 30 mM) and EC (30, 100, and 300 µM) for 48 h. (A) Total proteins (30 µg) of cellular lysate (cytosol) and medium were separated and analyzed on a 8% SDS-PAGE, and Western blot analysis was performed as described in Methods. Equal loading of cellular lysate was verified by probing the same blot for β-tubulin. (B) and (C) TSP-1 levels in cytosol (B) and medium (C) were determined by scanning densitometry of immunoblots. TSP-1 levels of cytosol and medium under 5.6 mM glucose without EC were set to 1. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 vs. 5.6 mM GLU without EC. †P < 0.05 for 30 mM GLU with EC vs. 30 mM GLU.

• 

  Fig. 3 The effects of epicatechin (EC) on high glucose-induced thrombospondin-1 (TSP-1) mRNA expression in rat aortic vascular smooth muscle cells (VSMCs). (A) VSMCs were incubated with glucose (GLU, 5.6 and 30 mM) or EC (30, 100, and 300 µM) for 24 h. The levels of TSP-1 mRNAs were measured by Northern blot analysis. Equal loading of sample (10 µg) was verified by the ribosomal 18S and 28S bands. (B) The levels of TSP-1 mRNAs expressed under 5.6 mM glucose without EC were set to 1. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 vs. 5.6 mM GLU without EC. †P < 0.05 for 30 mM GLU with EC vs. 30 mM GLU.

• 

  Fig. 4 The effects of epicatechin (EC) on the expression and activity of matrix metalloproteinase-2 (MMP-2) in rat aortic vascular smooth muscle cells (VSMCs). (A) VSMCs were incubated with glucose (GLU, 5.6 and 30 mM) with EC (30, 100, and 300 µM) for 24 h. The expressions of MMP-2 and GAPDH (as an internal control) mRNAs were measured by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. (B) The activity of MMP-2 was measured by gelatin zymography. Medium (25 µg) were separated and analyzed on an 8% polyacrylamide gel including gelatin (1 g/L) as described in Methods. (C) By scanning densitometry, level of MMP-2 activity under 5.6 mM GLU without EC was set to 1. Data are expressed as mean ± SD from three independent experiments. *, P < 0.05 vs. 5.6 mM GLU without EC. †, P < 0.05 for 30 mM GLU with EC vs. 30 mM GLU.

• 

  Fig. 5 The effects of epicatechin (EC) on the invasiveness of rat aortic vascular smooth muscle cells (VSMCs) using Matrigel-coated filters of Boyden chamber. VSMCs (2×105 cells/mL) were placed into the upper chamber of a Boyden chamber and were incubated with glucose (GLU, 5.6 and 30 mM) and EC (30, 100, and 300 µM) for 24 h. VSMCs migrated through Matrigel-coated filters to the lower surface were fixed with 2% paraformaldehyde and were stained with hematoxylin, and were counted for 5 high-power fields per well. The migrated VSMCs under 5.6 mM GLU without EC were set to 1. Data are expressed as mean ± SD from three independent experiments. *, P < 0.05 vs. 5.6 mM GLU without EC. †, P < 0.05 for 30 mM GLU with EC vs. 30 mM GLU.

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