J Korean Med Sci.  2012 Sep;27(9):1073-1078. 10.3346/jkms.2012.27.9.1073.

Early Sustained Injections of Erythropoietin Improve Angiogenesis and Restoration of Perfusion in the Ischemic Mouse Hindlimb

• Ahn S
• Min SK
• Min SI
• Suh JH
• Kim SJ
• Ha J

• KMID: 1782142
• DOI: http://doi.org/10.3346/jkms.2012.27.9.1073

Abstract

This study was conducted to investigate the effects of erythropoietin (Epo) on both acute and chronic limb ischemia (ALI and CLI) and to evaluate the differences in mechanisms according to the method of Epo administration. Hindlimb ischemia was made in BALB/c mice with femoral artery ligation. The mice were divided into four groups: Group 1 (control, no treatment), Group 2 (ALI, early multiple doses), Group 3 (ALI, early single high dose), Group 4 (CLI, late multiple doses). Blood flow ratio significantly increased in Group 2 in 4 weeks. Expression of pAkt and Erythropoietin receptor were significantly higher in Group 2 on postoperative day (POD) 7. The number of CD31- and vascular endothelial growth factor-positive cells were significantly higher in Group 2 on POD 7 and 56. Group 3 and 4 showed a tendency of higher cell counts than the control. The early sustained Epo was effective in improving blood flow through angiogenesis. In chronic phase, weekly multiple dosing of Epo induced angiogenesis, however, the blood flow ratio did not increase significantly. The results of this study suggest that Epo administration during the acute phase followed by maintenance for several days may be important for increasing blood flow and angiogenesis.

Keyword

Ischemia; Hindlimb; Erythropoietin; Angiogenesis

MeSH Terms

Acute Disease
Animals
Chronic Disease
Erythropoietin/*pharmacology
Hindlimb/*blood supply
Ischemia/*metabolism/pathology
Laser-Doppler Flowmetry
Male
Mice
Mice, Inbred BALB C
Neovascularization, Physiologic/drug effects
Proto-Oncogene Proteins c-akt/metabolism
Receptors, Erythropoietin/metabolism
Recombinant Proteins/pharmacology
Vascular Endothelial Growth Factor A/metabolism
Receptors, Erythropoietin
Recombinant Proteins
Vascular Endothelial Growth Factor A
Proto-Oncogene Proteins c-akt
Erythropoietin

Figure

• 

  Fig. 1 Study flowchart. The mice were divided into four groups: Group 1 (control, no treatment), Group 2 (ALI, early multiple doses of Epo), Group 3 (ALI, early single high dose of Epo), Group 4 (CLI, late multiple doses of Epo). Large arrows mean injection of 5,000 U/kg Epo and small arrows 1,000 U/kg Epo. Five mice died before the end of the experiment. Epo, erythropoietin; G1, Group 1; G2, Group 2; G3, Group 3; G4, Group 4; ALI, acute limb ischemia; CLI, chronic limb ischemia; POD, postoperative day.

• 

  Fig. 2 Representative results of Doppler blood flowmetry. (A) Ischemic/non-ischemic limb blood flow ratio value in mice. Group 2 shows improvement on the blood flow rate than other groups (*P < 0.048) after POD 28. (B) Laser Doppler blood flow (LDBF) pseudocolor image POD 56. Group 2 picture shows intact toes.

• 

  Fig. 3 Measurement of the CD31 and VEGF at POD 7 and POD 56. Number of the CD31 and VEGF positive cell count/HPF are significantly a higher in Group 2 than that of other groups at both POD 7 and POD 56. Group 3, 4 have a higher count of the CD31 and VEGF positive cell count than Group 1 at POD 56; *,†P < 0.05. VEGF, vascular endothelial growth factor; HPF, high power field.

• 

  Fig. 4 Identification of mobilized endothelial progenitor cells on POD 7 and POD 56. There are no statistical differences between the groups.

• 

  Fig. 5 Western blot result of pAkt and EopR at POD 7 and POD 56. The pAkt and EpoR are significantly higher in the Group 2 (*P < 0.05) at POD 7. However there are no statistical differences between the groups at POD 56. pAkt, phosphorylated protein kinase B; EpoR, erythropoietin receptor.

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