Korean Circ J.  2007 Oct;37(10):475-482. 10.4070/kcj.2007.37.10.475.

Inhibition of Neointima Formation by Anti-Vascular Endothelial Growth Factor and Receptor-1 Peptides in a Balloon-Injured Rat Carotid Artery

• Lee JM
• Moon KW
• Yoo KD
• Her SH
• Yoon HJ
• Jin SW
• Jeon DS
• Youn HJ
• Chung WS
• Seung KB
• Kim CM
• Kim JH
• Choi KB
• Hong SJ

• KMID: 2227048
• DOI: http://doi.org/10.4070/kcj.2007.37.10.475

Abstract

BACKGROUND AND OBJECTIVES: Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen. This study was undertaken to test the hypothesis that the neointima hyperplasia induced by a balloon injury is inhibited by blocking VEGF and VEGF receptor-1 (VEGFR-1) with anti-VEGF peptides. Materials and Methods: Anti-VEGF RRKRRR peptide (dRK6) and anti-VEGFR-1 peptide (anti-flt-1) were synthesized at Pohang University of Science and Technology, Korea. Male Sprague-Dawley rats, weighing 300-350 g, were subcutaneously injected 0.5 mg/kg of dRK6 or 0.5 mg/kg of anti-flt-1, dissolved in phosphate buffer solution, 2 days before induction of a carotid balloon-injury, and then daily in the same manner post carotid balloon injury for 2 weeks.
RESULTS
Neointima formation was suppressed in both the dRK6 and anti-flt-1 groups compared to that in the untreated controls at 2 weeks post carotid balloon-injury (neointimal area; control group 0.44+/-0.09 mm2, dRK6 group 0.25+/-0.05 mm2, anti-flt-1 group 0.19+/-0.05 mm2, p<0.01). Anti-flt-1 peptide and dRK6 reduced the numbers of proliferative bromodeoxyuridine-labeled cells in the neointima (control group 16.4+/-10.6%, dRK6 group 3.7+/-2.1%, anti-flt-1 group 5.9+/-3.4%, p<0.05). In addition, an inflammatory response, as determined by monocyte chemoattractant protein-1 and interleukin-6 upregulation, which was evident in the controls, was inhibited by both dRK6 and anti-flt-1.
CONCLUSION
This study suggests anti-vascular endothelial growth factor peptides can reduce the inflammation and neointima formation in balloon injured rat carotid arteries.

Keyword

Vascular endothelial growth factors; Vascular endothelial growth factor receptor-1; Peptides; Carotid artery injuries

MeSH Terms

Animals
Carotid Arteries*
Carotid Artery Injuries
Chemokine CCL2
Endothelial Growth Factors*
Gyeongsangbuk-do
Humans
Hyperplasia
Inflammation
Interleukin-6
Korea
Male
Neointima*
Peptides*
Rats*
Rats, Sprague-Dawley
Up-Regulation
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factors
Chemokine CCL2
Endothelial Growth Factors
Interleukin-6
Peptides
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factors

Figure

• 

  Fig. 1 Representative carotid arteries from the control (A and B), dRK6 (C and D) and anti-flt-1 groups (E and F) 14 days after balloon injury illustrating hematoxylin and eosin staining (A, C and E) and elastic fiber staining (B, D and F). dRK6 and anti-flt-1 both inhibited neointima formation 14 days post-injury. Magnification ×100. Bar indicates 100 µm. dRK6: anti-vascular endothelial growth factor RRKRRR peptide. RRKRRR: Arg-Arg-Lys-Arg-Arg-Arg.

• 

  Fig. 2 Neointimal area on day 14 after balloon injury. Neointimal areas were reduced in the dRK6 (n=5) and anti-flt-1 groups (n=5) vs. the control group (n=5). *means p<0.05, †p<0.01 by Bonferroni's test. dRK6: anti-vascular endothelial growth factor RRKRRR peptide. RRKRRR: Arg-Arg-Lys-Arg-Arg-Arg.

• 

  Fig. 3 BrDU staining of balloon-injured rat carotid arteries of the control (A and B), dRK6 (C and D) and anti-flt-1 groups (E and F) 14 days after balloon injury. Both dRK6 and anti-flt-1 reduced the numbers of BrDU positive cells, reflecting reduced neointimal cell proliferation 14 days post-injury. Magnification ×100 (A, C and E) and ×200 (B, D and F). Bar indicates 100 µm. BrDU: bromodeoxyuridine, dRK6: anti-vascular endothelial growth factor RRKRRR peptide, RRKRRR: Arg-Arg-Lys-Arg-Arg-Arg.

• 

  Fig. 4 Arterial cross sections taken 14 days post-injury were immunohistochemically stained for VEGF, VEGFR-1 (flt-1) and VEGFR-2 (flk-1). Strong VEGF and VEGFR-1 immunoreactivities in the media of the control group. dRK6 or anti-flt-1 treatments reduced VEGFR-1 immunoreactivities on day 14, but no changes were observed in the VEGF immunoreactivities. VEGFR-2 was expressed on the luminal side of the control group, but was absent in the dRK6 andanti-flt-1 groups. Magnification ×400. # indicates lumen. Bar indicates 100 µm. VEGF: vascular endothelial growth factor, VEGFR-1: VEGF receptor-1, VEGFR-2: VEGF receptor-2, BrDU: bromodeoxyuridine, dRK6: anti-VEGF RRKRRR peptide, RRKRRR: Arg-Arg-Lys-Arg-Arg-Arg.

• 

  Fig. 5 Carotid artery sections from the control, dRK6 and anti-flt1 groups 14 days post-injury stained with MCP-1 and IL-6. The immunoreactive MCP-1 and IL-6 in neointimal cells of the control group were attenuated by the administration of both dRK6 and anti-flt-1 peptides. Magnification ×400. Bar indicates 50 µm. dRK6: anti-vascular endothelial growth factor RRKRRR peptide, MCP-1: monocyte chemoattractive protein-1, IL-6: Interleukin-6, RRKRRR: Arg-Arg-Lys-Arg-Arg-Arg.

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