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Hypoxia pretreatment of bone marrow mesenchymal stem cells facilitates angiogenesis by improving the function of endothelial cells in diabetic rats with lower ischemia.

Liu J, Hao H, Xia L, Ti D, Huang H, Dong L, Tong C, Hou Q, Zhao Y, Liu H, Fu X, Han W - PLoS ONE (2015)

Bottom Line: Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI).CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days.The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, Beijing, China.

ABSTRACT
Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI). Mesenchymal stem cells (MSCs) are multipotent secretory cells that are suitable for clinical DLLI treatment, but their use has been hampered by poor survival after injection. Hypoxia can significantly enhance the capacity of MSCs to secrete angiogenic factors. We investigated transient hypoxia pretreatment of MSCs to facilitate revascularization in DLLI. Rat bone marrow MSCs (BM-MSCs) were cultured at different oxygen concentrations for varying time periods. The results indicated that transient pretreatment (5% O2, 48 h) not only increased the expression of VEGF-1α, ANG, HIF-1α and MMP-9 in BM-MSCs as assessed by real-time RT-PCR, but also increased the expression of Bcl-2 as determined by western blotting. The transplantation of pretreated BM-MSCs into rats with DLLI demonstrated accelerated vascular reconstruction when assayed by angiography and immunohistochemistry. CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days. The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle. Double immunofluorescence studies confirmed that the pretreated BM-MSCs promoted the proliferation and inhibited the apoptosis of endothelial cells. In vitro, pretreated BM-MSCs increased the migratory and tube forming capacity of endothelial cells (ECs). Hypoxia pretreatment of BM-MSCs significantly improved angiogenesis in response to tissue ischemia by ameliorating endothelial cell dysfunction and is a promising therapeutic treatment for DLLI.

No MeSH data available.


Related in: MedlinePlus

Hypoxia pretreatment promoted the function of BM-MSCs in ischemic muscle.(A) Hypoxia pretreatment of BM-MSCs increased the capacity for survival in ischemic muscle, compared to the normoxia group, (B) and the number of positive cells was measured. *, ** indicate P<0.05, 0.01 versus normoxia group, respectively. (C) The expression of VEGF-1α, MMP-9, and VEGFR was increased in hypoxia-pretreated BM-MSCs, and (D, E) the expression of pAKT was significantly enhanced, compared to the control group. * indicates P<0.05 versus control group.
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pone.0126715.g006: Hypoxia pretreatment promoted the function of BM-MSCs in ischemic muscle.(A) Hypoxia pretreatment of BM-MSCs increased the capacity for survival in ischemic muscle, compared to the normoxia group, (B) and the number of positive cells was measured. *, ** indicate P<0.05, 0.01 versus normoxia group, respectively. (C) The expression of VEGF-1α, MMP-9, and VEGFR was increased in hypoxia-pretreated BM-MSCs, and (D, E) the expression of pAKT was significantly enhanced, compared to the control group. * indicates P<0.05 versus control group.

Mentions: We confirmed that hypoxia pretreatment of BM-MSCs activated the expression of anti-apoptotic proteins in vitro. Based on this result, we analyzed the survival of BM-MSCs in vivo. The cell tracer results indicated that there were a similar number of cells in the three groups at 3 d. Thereafter, the survival of the hypoxia-pretreated BM-MSCs was higher than that of the normoxia group. Until day 14, approximately 5% of the hypoxia-pretreated BM-MSCs remained, whereas no surviving cells were observed in the normoxia group (Fig 6A and 6B). We further detected the survival of BM-MSCs in non-ischemic muscle, the results shows that BM-MSCs reduced survival ability compared with the ischemia group (S3 Fig). We also detected the expression of antigenic factors in the ischemic tissues. The results indicated that the expression of angiogenic factors was significantly increased in the hypoxia-pretreated BM-MSC group at days 3, 7, and 14 (Fig 6C). However, while the survival of BM-MSCs was enhanced by hypoxia pretreatment in vivo, we examined the expression of downstream target proteins mediated by proangiogenesis factors and found that the expression of pAKT (1:400, Cell Signaling) was upregulated and that the expression of AKT (1:600, Cell Signaling) exhibited no changes at day 7 (Fig 6D and 6E). Hypoxia pretreatment of BM-MSCs promoted the survival ability of the injected cells, increased the expression of angiogenic factors in ischemic tissue and activated the expression of angiogenesis-related signals.


Hypoxia pretreatment of bone marrow mesenchymal stem cells facilitates angiogenesis by improving the function of endothelial cells in diabetic rats with lower ischemia.

Liu J, Hao H, Xia L, Ti D, Huang H, Dong L, Tong C, Hou Q, Zhao Y, Liu H, Fu X, Han W - PLoS ONE (2015)

Hypoxia pretreatment promoted the function of BM-MSCs in ischemic muscle.(A) Hypoxia pretreatment of BM-MSCs increased the capacity for survival in ischemic muscle, compared to the normoxia group, (B) and the number of positive cells was measured. *, ** indicate P<0.05, 0.01 versus normoxia group, respectively. (C) The expression of VEGF-1α, MMP-9, and VEGFR was increased in hypoxia-pretreated BM-MSCs, and (D, E) the expression of pAKT was significantly enhanced, compared to the control group. * indicates P<0.05 versus control group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440823&req=5

pone.0126715.g006: Hypoxia pretreatment promoted the function of BM-MSCs in ischemic muscle.(A) Hypoxia pretreatment of BM-MSCs increased the capacity for survival in ischemic muscle, compared to the normoxia group, (B) and the number of positive cells was measured. *, ** indicate P<0.05, 0.01 versus normoxia group, respectively. (C) The expression of VEGF-1α, MMP-9, and VEGFR was increased in hypoxia-pretreated BM-MSCs, and (D, E) the expression of pAKT was significantly enhanced, compared to the control group. * indicates P<0.05 versus control group.
Mentions: We confirmed that hypoxia pretreatment of BM-MSCs activated the expression of anti-apoptotic proteins in vitro. Based on this result, we analyzed the survival of BM-MSCs in vivo. The cell tracer results indicated that there were a similar number of cells in the three groups at 3 d. Thereafter, the survival of the hypoxia-pretreated BM-MSCs was higher than that of the normoxia group. Until day 14, approximately 5% of the hypoxia-pretreated BM-MSCs remained, whereas no surviving cells were observed in the normoxia group (Fig 6A and 6B). We further detected the survival of BM-MSCs in non-ischemic muscle, the results shows that BM-MSCs reduced survival ability compared with the ischemia group (S3 Fig). We also detected the expression of antigenic factors in the ischemic tissues. The results indicated that the expression of angiogenic factors was significantly increased in the hypoxia-pretreated BM-MSC group at days 3, 7, and 14 (Fig 6C). However, while the survival of BM-MSCs was enhanced by hypoxia pretreatment in vivo, we examined the expression of downstream target proteins mediated by proangiogenesis factors and found that the expression of pAKT (1:400, Cell Signaling) was upregulated and that the expression of AKT (1:600, Cell Signaling) exhibited no changes at day 7 (Fig 6D and 6E). Hypoxia pretreatment of BM-MSCs promoted the survival ability of the injected cells, increased the expression of angiogenic factors in ischemic tissue and activated the expression of angiogenesis-related signals.

Bottom Line: Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI).CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days.The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, Beijing, China.

ABSTRACT
Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI). Mesenchymal stem cells (MSCs) are multipotent secretory cells that are suitable for clinical DLLI treatment, but their use has been hampered by poor survival after injection. Hypoxia can significantly enhance the capacity of MSCs to secrete angiogenic factors. We investigated transient hypoxia pretreatment of MSCs to facilitate revascularization in DLLI. Rat bone marrow MSCs (BM-MSCs) were cultured at different oxygen concentrations for varying time periods. The results indicated that transient pretreatment (5% O2, 48 h) not only increased the expression of VEGF-1α, ANG, HIF-1α and MMP-9 in BM-MSCs as assessed by real-time RT-PCR, but also increased the expression of Bcl-2 as determined by western blotting. The transplantation of pretreated BM-MSCs into rats with DLLI demonstrated accelerated vascular reconstruction when assayed by angiography and immunohistochemistry. CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days. The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle. Double immunofluorescence studies confirmed that the pretreated BM-MSCs promoted the proliferation and inhibited the apoptosis of endothelial cells. In vitro, pretreated BM-MSCs increased the migratory and tube forming capacity of endothelial cells (ECs). Hypoxia pretreatment of BM-MSCs significantly improved angiogenesis in response to tissue ischemia by ameliorating endothelial cell dysfunction and is a promising therapeutic treatment for DLLI.

No MeSH data available.


Related in: MedlinePlus