Bone marrow mononuclear cell transplantation promotes therapeutic angiogenesis via upregulation of the VEGF-VEGFR2 signaling pathway in a rat model of vascular dementia

Abstract

Bone marrow mononuclear cells (BMMNCs) are important for angiogenesis after stroke. We investigated the effects of BMMNCs on cognitive function, angiogenesis, and the vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of vascular dementia. We transplanted BMMNCs into rats that had undergone permanent bilateral occlusion of the common carotid arteries (2VO) and observed their migration in vivo. On day 28, we assessed cognitive function with the Morris Water Maze test and examined vascular density and white matter damage within the corpus striatum by staining with fluorescein lycopersicon esculentum (tomato) lectin or Luxol fast blue. We evaluated expression of VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in the ischemic hemisphere by Western blot analysis on day 7 after cell transplantation. Contribution of the VEGF-VEGFR2 signaling pathway was confirmed by using VEGFR2 inhibitor SU5416. BMMNCs penetrated the blood-brain barrier and reached the ischemic cortex and white matter or incorporated into vascular walls of 2VO rats. BMMNC-treated 2VO rats had better learning and memory, higher vascular density, and less white matter damage than did vehicle-treated rats. The beneficial effects of BMMNCs were abolished by pretreatment of rats with SU5416. Protein expression of VEGF and phosphorylated Raf1 and ERK1/2 was also significantly increased by BMMNC treatment, but this upregulation was reversed by SU5416. BMMNCs can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery in 2VO rats. The angiogenic effect may result from upregulation of the VEGF-VEGFR2 signaling pathway.

Keywords: Angiogenesis; Bone marrow mononuclear cells; Cell transplantation; VEGF–VEGFR2 signaling pathway; Vascular dementia.

Figure 1

BMMNC transplantation alleviates neuronal damage in the CA1 subfield of hippocampus but does not affect body weight changes. (A–D) Neuronal damage in the hippocampal CA1 region was greater in vehicle-treated 2VO rats than in vehicle-treated sham rats on day 7 post-treatment. BMMNC transplantation mitigated the neuronal loss and degeneration in the CA1 region. Scale bars: A = 200 μm, B = 100 μm, C = 20 μm, D=30 μm. n=5/group. (E–F) Quantification showed that more viable neurons and Fluoro-Jade B-positive neurons were present in the CA1 region of the 2VO+BMMNC group than in that of the 2VO+vehicle group. *p<0.05 vs. vehicle-treated group. (G) Changes in rat body weight over the course of the experiment (n=18/group). Although BMMNC treatment promoted recovery of body weight, no significant difference was found among the four 2VO groups. p>0.05.

Figure 2

In vivo migration of BMMNCs in rats that underwent 2VO. (A) Immunofluorescence staining for BrdU-labeled cells revealed that BMMNCs (red) migrated to both cortex (7 days) and white matter (corpus striatum area, 1, 3, and 14 days). (B) Double Immunofluorescence staining showed that BMMNCs (red) can be detected 2 weeks after transplantation in the area of corpus striatum. (C) The distribution of BMMNCs in the area of frontoparietal cortex. Most cells were incorporated into the vascular wall by day 14 after cell transplantation. Scale bars = 50 μm. n=3 at each time point. (D) Quantification showed that more vascular BrdU-positive cells were present in corpus striatum than in cortex. *p<0.05.

Figure 3

BMMNCs improve learning and memory at day 28 after treatment. (A) Latency to find the platform in the Morris Water Maze test. Rats that had undergone 2VO exhibited worse performance than did the sham-operated rats. Treatment of 2VO rats with BMMNCs decreased the latency to find the platform (improved learning) compared to vehicle treatment. 2VO rats infused with SU5416 (2VO+SU5416) had poorer performance than did the 2VO+vehicle and 2VO+ SU5416+BMMNC groups. *p<0.01 vs. the sham groups; #p<0.01 vs. the other three 2VO groups; †p<0.05 vs. 2VO+vehicle group. (B) There was no difference in swimming speed among the six groups during the five sessions. (C) Quantification of time spent in the target zone during the probe trial indicated that sham groups spent more time in the correct quadrant than did the 2VO groups. The 2VO+BMMNC group exhibited better performance than did the other three 2VO groups. The 2VO+SU5416 group spent less time in the target zone than did the 2VO+SU5416+BMMNC group. *p<0.01 vs. sham groups; #p<0.05 vs. the other three 2VO groups; †p<0.05 vs. 2VO+SU5416+BMMNC group. (D) Sham groups had more crossings of the annulus than did the 2VO groups. 2VO rats treated with BMMNCs had more crossings than did vehicle-treated, SU5416+BMMNC-treated, or SU5416-treated 2VO rats. *p<0.05 vs. sham groups; #p<0.05 vs. the other three 2VO groups. n=12/group.

Figure 4

BMMNCs facilitate angiogenesis in 2VO rats. (A) Immunofluorescence staining of blood vessels (green) with fluorescein lycopersicon esculentum (tomato) lectin in corpus striatum on day 28 after administration of BMMNCs. (B) Quantification showed that 2VO groups had significantly higher vascular density than did sham groups. In addition, the vascular density of the 2VO+BMMNC group was significantly higher than that of the other three 2VO groups. *p<0.01 vs. sham groups. #p<0.01 vs. the other three 2VO groups. Scale bar = 50 μm. n=6/group.

Figure 5

BMMNCs decrease white matter lesions in mid corpus callosum and corpus striatum of rats with 2VO. (A) Luxol fast blue staining of the middle of the corpus callosum on day 28 after treatment with BMMNCs. (B) Luxol fast blue staining of the corpus striatum on day 28 after treatment with BMMNCs. Scale bars in A and B = 30 μm. (C, D) Quantification of Luxol fast blue staining of the mid corpus callosum and corpus striatum shows that 2VO rats had less myelin than did sham groups. BMMNC transplantation significantly increased Luxol fast blue staining, but the effect was neutralized by SU5416. *p<0.01 vs. sham groups. #p<0.01 vs. the other three 2VO groups. n=6/group.

Figure 6

BMMNCs increase the expression of VEGF, phosphorylated Raf1 (p-Raf1), and phosphorylated ERK1/2 (p-ERK) in 2VO rats. (A) Western blot analysis of VEGF, p-Raf1, total (t) Raf1, p-ERK, and t-ERK1/2 on day 7 after BMMNC transplantation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Quantification of band densities showed that VEGF expression was significantly greater in the 2VO group treated with BMMNCs than in the other three 2VO groups. *p<0.05 vs. sham groups; #p<0.05 vs. the other three 2VO groups. (C, D) Quantification of band densities as a ratio of phosphorylated protein/total protein showed that BMMNC treatment significantly increased Raf1 and ERK1/2 phosphorylation compared to that in the two sham groups and the other three 2VO groups. *p<0.05 vs. sham groups; #p<0.05 vs. the other three 2VO groups. n=6 per group.