Exogenous endothelial progenitor cells reached the deficient region of acute cerebral ischemia rats to improve functional recovery via Bcl-2

Abstract

Background: As discovered in our previous study, autologous endothelial progenitor cells (EPCs) protect against acute focal ischemia rat via the promotion of angiogenesis. However, it is unknown whether the EPCs that reached the deficient region were transplanted ones or the products of other auto-conversion cells they had promoted. This study aimed to gather direct evidence for determining if exogenous transplanted EPCs directly participate in angiogenesis in ischemic areas and attempted to clarify the related mechanism.

Methods: First, EPCs were extracted in vitro from male rats, which were characterized by uptake of fluorescently labeled acetylated low-density lipoprotein (ac-LDL) intake and Ulex europaeus agglutinin (UEA-1) and subsequently introduced to middle cerebral artery occlusion (MCAO) female rats for 7 days after ischemia surgery. The EPC-treated animals received approximately 1×10⁶ cells, while the control animals received phosphate buffered saline (PBS). The animals behavior function recovery were by a rotarod (TOR) test, while infarct volume was assessed by brain magnetic resonance imaging (MRI). CD31 antibody was used to determine the presence of EPCs in the ischemic zone, and sex-determining region Y (SRY) gene in-situ hybridization (ISH) traced the EPC process. In addition, immunohistochemistry and Western blot were used to assess B-cell lymphoma 2 (Bcl-2) expression in the ischemic brain.

Results: Behavior tests and MRI of all ischemic stroke groups on postoperative day 14 indicated that EPCs were more effective in behavior function recovery and reducing infarct volume and gliosis status than the control group. Cluster of differentiation (CD31) immunofluorescent staining and SRY gene ISH demonstrated that EPCs yielded a better outcome in both angiogenesis and exogenous cell homing status. We also observed increased Bcl-2 distribution and higher plasma Bcl-2 levels in the EPC-treated group compared to the control group.

Conclusions: Our results provide direct evidence that exogenous EPCs can participate in angiogenesis to improve neurological outcome and revascularization directly after stroke, with Bcl-2 playing an important role in this process.

Keywords: Bcl-2; Stroke; angiogenesis; behavior; endothelial progenitor cells (EPCs).

Figure 1

Validation of male rat-derived EPC. Bone-marrow derived MNCs differentiated into endothelial progenitor cells (14 days in vitro). (A) EPC uptake of FITC-UEA-I (green, 400×). (B) The majority of adherent cells were both Dil-ac-LDL and lectin positive (yellow overlap, 400×). (C) EPC uptake of Dil-ac-LDL (red, 400×). EPC, endothelial progenitor cell; MNCs, mononuclear cells.

Figure 2

Rotarod test of the sham, PBS, and EPC group on days 0, 1, 3, 7, and 14. The symbol (*) indicates the significant differences among groups at the same time point. EPC, endothelial progenitor cell; PBS, phosphate buffered saline.

Figure 3

MRI of MCAO groups on postoperative day 14. (A,B,C) Brain MRI findings evaluated ischemic lesion volumes and gliosis status: (A) PBS group; (B) EPC group; (C) sham group. The arrow represents the ischemic volume in the corresponding cerebral areas. (D,E) Ischemic volume analysis: (D) cerebral cortex T1W signal analysis. The results showed that the volume was higher in the PBS group than in the EPC and sham groups (*, P<0.05); (E) cerebral cortex T2W signal analysis yielded the same results as the T1W (*, P<0.05). MRI, magnetic resonance imaging; MCAO, middle cerebral artery occlusion; EPC, endothelial progenitor cell; PBS, phosphate buffered saline.

Figure 4

ISH test showing the SRY gene-positive cell count in female rats. (A) Sham and PBS groups (black arrows show the SRY gene-negative endothelial cells); (B) EPC group (red arrows show SRY gene-positive endothelial cells). ISH, in-situ hybridization; EPC, endothelial progenitor cell.

Figure 5

CD31-positive cell immunofluorescent staining. The red area represents the CD31-positive cell membrane, and the blue area represents the cell nucleus. (A) Sham group; (B) PBS group; (C) EPC group; (D) vascular density analysis. The difference between all three groups was significant, and the EPC group had the highest number of CD31-positive cells (*, P<0.05). EPC, endothelial progenitor cell; PBS, phosphate buffered saline.

Figure 6

Immunohistochemistry analysis of EPC transplantation on expression levels of Bcl-2. (A,B,C) Representative micrographs (×200) of brain tissue in the sham (A), PBS-treated (B) and EPC-treated (C) groups. (D) Quantitative analysis of Bcl-2-positive cells in the sham, PBS-treated and EPC-treated groups. The results are expressed as mean ± SD. The level of Bcl-2 in the EPC group was higher than that in the PBS- and sham-treated groups (*, P<0.05). EPC, endothelial progenitor cell; PBS, phosphate buffered saline.

Figure 7

Western blot analysis of EPC transplantation on expression levels of Bcl-2. Western blot analysis with β-actin used as an internal control. (A) The expression levels of Bcl-2 in the sham, PBS-treated and EPC-treated groups (n=6). (B) Comparison between the PBS- and EPC-treated groups revealed that EPCs elevated the levels of Bcl-2 in the brain; *, P<0.05. EPC, endothelial progenitor cell; PBS, phosphate buffered saline.