Recombinant Human Erythropoietin Augments Neovascularization Responses in a Neonatal Rat Model of Premature Brain Damage by Phosphatidylinositol 3 Kinase/Akt Pathway

Abstract

Background: Recombinant human-erythropoietin (rh-EPO) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti-inflammation. In the present study, we found that the rh-EPO was related to the promotion of neovascularization. Our aim was to investigate whether rh-EPO augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway.

Methods: Postnatal day 5 (PD5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. All the rat pups were randomized into five groups as follows: (1) control group; (2) hypoxia-ischemic (HI) group; (3) HI + LY294002 group; (4) HI + rh-EPO group; and (5) HI + rh-EPO + LY294002 group. The phospho-Akt protein was tested 90 min after the whole operation, and CD34, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor (VEGF) were also tested 2 days after the whole operation.

Results: In the hypoxic and ischemic zone of the premature rat brain, the rh-EPO induced CD34+ cells to immigrate to the HI brain zone (P < 0.05) and also upregulated the VEGFR2 protein expression (P < 0.05) and VEGF mRNA level (P < 0.05) through the PI3K/Akt (P < 0.05) signaling pathway when compared with other groups.

Conclusions: The rh-EPO treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the PI3K/Akt signaling pathway. Besides, the endogenous EPO may exist in the HI zone of rat brain and also has neovascularization function through the PI3K/Akt signaling pathway.

Figure 1

Expressions of p-Akt, CD34, and VEGFR2 in the right white matter of premature brain were analyzed by Western blot analysis. (a) Representative bands and bar graph showing expression of p-Akt 90 min after the whole operation. (b) Representative bands and bar graph showing expression of CD34 2 days after the whole operation. (c) Representative bands and bar graph showing expression of VEGFR2 2 days after the whole operation. Group IV showing significant difference from other groups (P < 0.05); Group III showing significant difference from Groups IV, V, and II (P < 0.05); Group II showing significant difference from Group I (P < 0.05). VEGFR2: Vascular endothelial growth factor receptor 2.

Figure 2

Expressions of CD34⁺ cells and VEGF in the right white matter of premature brain 2 days after the whole operation. (a) Immunofluorescence staining for CD34⁺ cells (red: CD34; blue: nucleus). (b) A histogram showing quantification difference in the number of CD34⁺ cells. (c) VEGF mRNA level was analyzed by qRT-PCR. Group IV showing significant difference from other groups (P < 0.05); Group III showing significant difference from Groups IV, V, and II (P < 0.05); Group II showing significant difference from Group I (P < 0.05). GAPDH: glyceraldehyde-3-phosphate dehydrogenase; VEGF: Vascular endothelial growth factor; qRT-PCR: Quantitative real-time-polymerase chain reaction.