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

• Kim TK
• Kim YJ
• Park CS
• Park HJ
• Kim DB
• Jang SW
• Kim PJ
• Jung HO
• Baek SH
• Seung KB
• Choi KB

• KMID: 2225822
• DOI: http://doi.org/10.4070/kcj.2008.38.4.212

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.

Keyword

Radiation therapy; Matrix metalloprotease-2; Carotid artery injuries

MeSH Terms

Animals
Blotting, Western
Carotid Arteries
Carotid Artery Injuries
Carotid Artery, Common
Constriction, Pathologic
Gelatin
Hyperplasia
Muscle, Smooth, Vascular
Peptide Hydrolases
Rats
Rats, Sprague-Dawley
Gelatin
Peptide Hydrolases

Figure

• 

  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.

Reference

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