Korean Circ J.  2010 Nov;40(11):552-557. 10.4070/kcj.2010.40.11.552.

Tumor Suppressor Serine/Threonine Kinase LKB1 Expression, Not Kinase Activity, Increased in the Vascular Smooth Muscle Cells and Neointima in the Rat Carotid Artery Injury Model

• Jeong JO
• Kim JH
• Ahn KT
• Park HS
• Jang WI
• Park JH
• Lee JH
• Choi SW
• Kim JM
• Seong IW

• KMID: 1826173
• DOI: http://doi.org/10.4070/kcj.2010.40.11.552

Abstract

BACKGROUND AND OBJECTIVES
Vascular smooth muscle cell (VSMC) proliferation is responsible for the restenosis of previously inserted coronary stents. Angiotensin II (Ang II) is known to regulate VSMC proliferation. LKB1, a serine/threonine kinase, interacts with the p53 pathway and acts as a tumor suppressor.
MATERIALS AND METHODS
We assessed the association of Ang II and the expression of LKB1 in primary cultured murine VSMCs and neointima of the Sprague Dawley rat carotid artery injury model. We created carotid balloon injuries and harvested the injured carotid arteries 14 days after the procedure.
RESULTS
Ang II increased LKB1 expression in a time-dependent manner and peaked at an Ang II concentration of 10(-7) mole/L in VSMCs. In the animal experiment, neointima was markedly increased after balloon injury compared to the control group. Immunohistochemical studies showed that LKB1 expression increased according to neointima thickness. Ang II augmented LKB1 expression after the injury. Western blot analysis of LKB1 with carotid artery lysate revealed the same pattern as LKB1 immunohistochemistry. Increased LKB1 expression started at 5 days after the balloon injury, and peaked at 14 days after the injury. Although LKB1 expression was increased after the injury, LKB1 kinase activity was not increased. Ang II or balloon-injury increased the expression of LKB1 although the LKB1 activity was reduced.
CONCLUSION
Ang II increased LKB1 expression in VSMCs and neointima. These findings were not kinase dependant.

Keyword

Angiotensin II; LKB1 protein, rat; Coronary restenosis

MeSH Terms

Angiotensin II
Animal Experimentation
Animals
Blotting, Western
Carotid Arteries
Carotid Artery Injuries
Coronary Restenosis
Immunohistochemistry
Muscle, Smooth, Vascular
Neointima
Phosphotransferases
Protein-Serine-Threonine Kinases
Rats
Stents
Angiotensin II
Phosphotransferases
Protein-Serine-Threonine Kinases

Figure

• 

  Fig. 1 A: VSMCs were growth arrested for 24 hours and treated with Ang II for 48 hours. Cells were harvested in sodium dodecyl sulfate sample buffer. Western blot analysis was performed with anti-LKB1 antibody. The results are representative of 3 experiments. Ang II increases serine/threonine kinase LKB1 in rat VSMCs. B: induction of LKB1 by Ang II in VSMCs. Quiescent VSMCs were maintained under control conditions in the presence of the indicated concentration of Ang II for 48 hours. Induction of LKB1 by Ang II peaked at an Ang II concentration of 10-7 mole/L. C: time response of LKB1 by Ang II. Quiescent VSMCs were maintained in the presence of Ang II (100 nM) for the indicated times. Induction of LKB1 peaked at 48 hours. TNF-α: tumor necrosis factor alpha, Ang II: angiotensin II, VSMCs: vascular smooth muscle cells.

• 

  Fig. 2 A, B and C: representative microphotographs of H&E staining in balloon-injured carotid artery at day 14 (200×, n=3/each group). A: sham operation. B: balloon-injured carotid artery. C: balloon-injured and Ang II infusion via a miniosmotic pump. A neointima was found 2 weeks after carotid artery balloon injury. A marked neointima was found after balloon injury (*). D, E and F: representative microphotographs of LKB1 immunohistochemistry stain in balloon-injured carotid artery at day 14 (400×, n=3/each group). D: sham operation. E: balloon-injured carotid artery. F: balloon-injured and Ang II infusion via a miniosmotic pump. LKB1 expression was increased after carotid artery balloon injury. LKB1 expression was more greatly increased by Ang II after carotid artery balloon injury. Expression of LKB1 was graded according to the following scale by two investigators blinded to the experimental conditions: negative (0), mild staining (1), moderate staining (2), and intense staining (3). I: representative western blot of the LKB1 in balloon-injured carotid artery at day 14 (n=3/each group). The carotid artery lysates containing an equal amount of protein from each condition were analyzed by Western blotting for LKB1 using specific antibodies. Western blot of LKB1 expression was similar to immunohistochemistry. LKB1 expression increased after carotid artery balloon injury. LKB1 expression was more greatly increased by Ang II after carotid artery balloon injury.

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  Fig. 3 Representative serial LKB1 staining showed increased LKB1 expression started 5 days after the balloon injury and peaked 14 days after the injury according to neointima formation (n=3/each day).

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  Fig. 4 A: LKB1 kinase activity was increased until 1 day after carotid artery balloon injury and then decreased (n=4/each time). B: western blot of LKB1 expression was not correlated with LKB1 kinase activity in balloon-injured carotid arteries.

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