| Backround and ObjectiveStroke is one of the leading causes of disability and death worldwide. Neurons were died from ischemia and hypoxia after stroke, so the timely reconstruction of revascularization in ischemic region is a key treatment for cerebral ischemic injury. Animal experiments and clinical studies showed that compensatory angiogenesis was increased after stroke. They could increase the blood perfusion of cerebral ischemia and limit the ischemic penumbra extension. Recently, most studies have shown that bone marrow mesenchymal stem cells (MSCs) transplantation could promote angiogenesis in ischemic zones and improve the recovery of impaired neurological functions. However, the mechanisms are unclear. Some researches have proved that Notch signaling pathway plays an important role in embryonic blood vessels development and tumor angiogenesis. But whether Notch involved in angiogenesis after cerebral ischemia is still unknown. In this study, we will explore the role of Notch signaling pathway in angiogenesis after stroke, which would provide theoretical basis for clinical application of MSCs transplantation in the treatment of stroke.Methods1. SD rat MSCs were isolated, cultured and purified by adherence screening method, and the forth generation MSCs were collected for transplantation experiments.2. The rats were divided into MSCs transplantation group, ischemic control group and nomal group randomly. Middle cerebral artery occlusion (MCAo) models were produced by obstructing MCA using a single strand nylon thread, and the nomal group rats were not operated. The right middle cerebral artery occlusion (MCAo) model MSCs were administrated at 24 hours after MCAo in transplantation group, while the same volume of PBS was injected in ischemic control group. The rats were killed at 1, 3, 7, 14 and 21 days after MCAo, and the brain tissues were extracted for examine. 3. The transplanted MSCs were identified and counted in the ischemic and contralateral zone by immunofluorescence staining.4. The expression ofⅧfactor was examined in frozen tissue sections by immunofluorescence staining, and the microvessels in ischemic cortex of each group were counted.5. The expression of VEGF in ischemic cortex was examined by immunofluor- escence staining and RT-PCR method in each group.6. Brain tissue frozen sections of each group were stained for Notch1 andⅧfactor by double immunofluorescence method, and the Notch1-positive microvessels in ischemic cortex were counted. The expressions of Notch1 and Hes1 in ischemic cortex were detected by Western Blot and RT-PCR method.Results:1. The forth passage of MSCs shows a fibroblast-like shape and most cells expressed CD29 instead of CD34 by immunofluorescent staining.2. Twenty-four hours later, the transplanted MSCs can be detected mainly in the ischemic zone. The amount of MSCs in ischemic area was significant higher than in contralateral area (7.96±1.28 vs. 3.07±0.77, n = 5, P<0.05). These suggested that the transplanted MSCs can migrate to the ischemia zone through the femoral vein graft in a short time.3. The immunofluorescent staining results showed a significant increase of microvessels in ischemic cortex in MSCs transplantation group compared with control group (3.96±0.33 vs. 2.85±0.51 at 1day post-transplantation; 4.68±0.64 vs. 4.47±0.44 at 3 days; 9.26±1.62 vs. 5.63±0.69 at 7 days; 8.88±1.34 vs.4.09±0.44 at 14 days and 6.98±1.54 vs. 3.75±0.72 at 21 days, P<0.05, respectively). In nomal group, the quantity of microvessels was 2.79±0.21. The double-immunofluorescent staining results confirmed a part ofⅧfactor-positive cells expressed CM-DiI also. These data suggested that MSCs transplantation could increase the number of microvessels in cerebral cortex, and some vascular endothelial cells differentiated from MSCs.4. The quantities of VEGF-positive cells in ischemic cortex of MSCs transplantation group was significantly higher than that of control group (6.98±0.54 vs. 3.66± 0.55 at 1day post-transplantation; 9.15±1.13 vs. 4.62±0.32 at 3 days; 11.14±0.98 vs. 5.88±0.62 at 7 days; 9.13±0.35 vs. 5.11±0.26 at 14 days and 8.85±0.53 vs. 3.99±0.32 at 21 days, P<0.05, respectively). In nomal group, the quantity of microvessels was 2.46±0.24. Furthermore, the expression of VEGF165mRNA was increased in transplantation group compared with control group and nomal group. The results indicated that MSCs treatment could increase the expression of VEGF protein and VEGF165mRNA of ischemic cortex.5. The quantities of Notch1-positive microvessels in ischemic cortex were significantly higher in MSCs transplantation group compared with control rats (3.30±0.26 vs. 2.57±0.24 at 1day post-transplantation; 3.73±0.25 vs. 1.78±0.28 at 3 days; 6.85±0.38 vs. 0.61±0.14 at 7 days; 3.78±0.59 vs. 0.05±0.05 at 14 days and 0.49±0.25 vs. 0.02±0.02 at 21 days, P<0.05, respectively). In nomal group, no Notch1-positive microvessel was detected. The expression of Notch1 and Hes1 were significantly higher in MSCs transplantation group compared with ischemia control group and sham operation group. In all, the results showed that MSC transplantation could promote Notch1 protein, Notch1mRNA and Hes1mRNA expressions of ischemic cortex endothelial cells.Conclusion:1. The transplanted MSCs have the tropism of migration to ischemic zones via femoral vein injection. They can participate in the formation of new blood vessels directly and promote angiogenesis in ischemic cortex.2. MSCs transplantation could activate Notch signaling pathway of endothelial cells by promoting the expression level of VEGF in ischemic cortex, and promote the formation of new blood vessels. The Notch signaling pathway was involved in the regulation of angiogenesis after MCAo. |
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