Effect of High Dose External Irradiation on the Matrix Metalloprotease-2 Expression in a Rat Carotid Artery Injury Model

Tae Kyoon Kim; Yong Joo Kim; Chan Seok Park; Hun-Jun Park; Dong-Bin Kim; Sung-Won Jang; Pum-Joon Kim; Hae-Ok Jung; Sang Hong Baek; Ki Bae Seung; Kyu-Bo Choi

doi:10.4070/kcj.2008.38.4.212

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Kim, Kim, Park, Park, Kim, Jang, Kim, Jung, Baek, Seung, and Choi: Effect of High Dose External Irradiation on the Matrix Metalloprotease-2 Expression in a Rat Carotid Artery Injury Model

Original Article

Korean Circulation J 2008;38(4):212-219.

Published online: 20 January 2008

DOI: https://doi.org/10.4070/kcj.2008.38.4.212

Effect of High Dose External Irradiation on the Matrix Metalloprotease-2 Expression in a Rat Carotid Artery Injury Model

Tae Kyoon Kim, MD, Yong Joo Kim, MD, Chan Seok Park, MD, Hun-Jun Park, MD, Dong-Bin Kim, MD, Sung-Won Jang, MD, Pum-Joon Kim, MD, Hae-Ok Jung, MD, Sang Hong Baek, MD, Ki Bae Seung, MD, Kyu-Bo Choi, MD

Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea

Correspondence: Sang Hong Baek, MD, Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea Tel: 82-2-590-2075, Fax: 82-2-591-1506 E-mail: whitesh@catholic.ac.kr

Received 29 May 2007 Revised 5 November 2007 Accepted 26 November 2007

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background and Objectives

Remodeling of the injured arterial wall is dependent on the action of several extracellular proteases, including matrix metalloprotease-2 (MMP-2), and this protein is associated with the migration of vascular smooth muscle cells. The effect of a high dose of external irradiation (20 Gy) on the MMP-2 expression in neointimal hyperplasia is not known.

Materials and Methods

Sprague-Dawley rats were subjected to balloon injury to the common carotid artery. At 24 hours after injury, 20 Gy external irradiation was done for the irradiated group (n=25) and this was not done for the control group (n=25). The percent area stenosis, the maximal intimal thickness, the intima/media area ratio on H-E staining and the MMP-2 positivity on the immunohistochemical staining were measured. Western blotting and a gelatin zymogram for determining the MMP-2 protein expression were also performed after the injury.

Results

The parameters of neointimal hyperplasia such as the percent area stenosis, the maximal intimal thickness and the intima/media area ratio were 40.2±12.1%, 0.30±0.12 mm and 1.27±0.32, respectively, at 14 days after injury, and these parameters were maintained as a hyperplastic state at 28 days after injury in the control group. There was undetectable neointimal hyperplasia in the irradiated group compared with the control group (p<0.01). Western blotting demonstrated an increase in the MMP-2 protein level beginning 2 to 4 days after injury in the control group, but there was only a transient increase in the MMP-2 level at day 2 after injury in the irradiated group. The gelatin zymogram and immunohistochemical staining also showed the expression of MMP-2 in the control group, but not in the irradiated group.

Conclusion

These findings suggest the suppressed expression of MMP-2 is associated with reduced neointimal hyperplasia in the balloon injury-rat model.

Keywords: Radiation therapy, Matrix metalloprotease-2, Carotid artery injuries

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Fig. 1.

Photomicrographs from rat carotid artery stained with hematoxylin-eosin(×40). There is a marked increasing neointimal formation at 8 days (A), 14 days (C), and 28 days (E) after injury in control group. There is a nearly absence of neointimal area at 8 days (B), 14 days (D), and 28 days (F) after injury in irradiated group. Arrows indicate borderline of neointima.

Fig. 2.

Effect of 20 Gy external irradiation on neointimal hyperplasia from rat carotid artery. A: percent area stenosis is nearly completely inhibited in irradiated group compared with control group at 14 days and 28 days after injury (p<0.01). B: intima/media ratio is also significantly inhibited in irradiated group compared with control group at 14 days and 28 days after injury (p<0.01). ∗Compared with control group at 14 days after injury (p<0.01),^†Compared with control group at 28 days after injury (p<0.01). CTL: control group, RT: irradiated group.

Fig. 3.

Effect of 20 Gy external irradiation on western blot for MMP-2 in the balloon-injured rat carotid artery. A: representative western blot for MMP-2. Three micrograms of total protein extracted from a single vessel at each time point was loaded in each lane. B: graph of combined data for MMP-2 protein levels. The ratio is the representative O.D. data compared with the O.D. of sham operation. Data represent results from five separate determinations. ∗Statistically significant from sham operation group (p<0.05). PC: positive control for MMP-2 (lung tissue of rat), S: sham operation group, CTL: control group, RT: irradiated group, O.D.: optical density, MMP-2: matrix metalloprotease-2.

Fig. 4.

Effect of 20 Gy external irradiation on gelatin zymogram in the balloon-injured rat carotid artery. A: representative gelatin zymogram for latent and active MMP-2. Three micrograms of total protein extracted from a single vessel at each time point was loaded in each lane. B: relative activation of MMP-2 as a function of time after artery injury. Graph of combined data for MMP-2 protein levels. Data represent results from five separate determinations. ∗Statistically significant from sham operation group (p<0.05). ^†Statistically significant from unirradiated group (p<0.05). S: sham operation group, PC: positive control for MMP-2 (media of concanavalin-treated HT 1080 cell), CTL: control group, RT: irradiated group, MMP-2: matrix metalloprotease-2.

Fig. 5.

Photomicrographs from rat carotid arteries stained with immunohistochemistry of MMP-2. MMP-2 positivity is found in neointima area at 14 days (A) and 28 days (C) after balloon injury in control group. Cells with dark brown staining in the intima indicate MMP-2 positive cells. There is no neointimal area no MMP-2 positive cell at 14 days (B) and 28 days (D) after balloon injury in irradiated group. MMP-2: matrix metalloprotease-2.

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