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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 of BM-MSCs enhanced the capacity for angiogenesis and reduced apoptosis in HUVECs.(A) Hypoxia pretreatment of BM-MSCs enhanced the capacity for migration by HUVECs after 12 h, as compared to the control group; the efficiency of cell migration was detected. * indicates P<0.05 versus control group. (B) Hypoxia pretreatment of BM-MSCs increased the ability for tubular formation by HUVECs after 24 h, as compared to the control group; the average length of the tubular formations was calculated. * indicates P<0.05 versus control group. (C,D) H2O2-treated HUVECs exhibited high expression of caspase-3. The CM from hypoxia-pretreated BM-MSCs depressed the expression of caspase-3 and enhanced the expression of Bcl-2. * indicates P<0.05 versus control group.
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pone.0126715.g002: Hypoxia pretreatment of BM-MSCs enhanced the capacity for angiogenesis and reduced apoptosis in HUVECs.(A) Hypoxia pretreatment of BM-MSCs enhanced the capacity for migration by HUVECs after 12 h, as compared to the control group; the efficiency of cell migration was detected. * indicates P<0.05 versus control group. (B) Hypoxia pretreatment of BM-MSCs increased the ability for tubular formation by HUVECs after 24 h, as compared to the control group; the average length of the tubular formations was calculated. * indicates P<0.05 versus control group. (C,D) H2O2-treated HUVECs exhibited high expression of caspase-3. The CM from hypoxia-pretreated BM-MSCs depressed the expression of caspase-3 and enhanced the expression of Bcl-2. * indicates P<0.05 versus control group.

Mentions: Furthermore, we observed the effects of hypoxia pretreatment of BM-MSCs on ECs in vitro. Conditioned medium (CM) was prepared from hypoxia-pretreated BM-MSCs that were concentrated 10 times and were used to evaluate the paracrine effect of BM-MSCs on HUVECs as well as the function of CM in vivo (S2 Fig). The results indicated that non-concentrated CM from normoxic BM-MSCs (N-CM) increased the capacity of migration and tube formation on HUVECs as compared to the control group, and this capacity was significantly increased after treatment with CM from hypoxia-pretreated BM-MSCs (H-CM) (Fig 2A and 2B). Increasing the anti-apoptotic function of ECs is important for angiogenesis. Therefore, we investigated the effects of CM on HUVECs apoptosis. The results indicated that H-CM downregulated the expression of caspase-3, which is induced at 4 h by H2O2, and upregulated the expression of Bcl-2 (Fig 2C and 2D). Therefore, H-CM improved the function of HUVECs in vitro.


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 of BM-MSCs enhanced the capacity for angiogenesis and reduced apoptosis in HUVECs.(A) Hypoxia pretreatment of BM-MSCs enhanced the capacity for migration by HUVECs after 12 h, as compared to the control group; the efficiency of cell migration was detected. * indicates P<0.05 versus control group. (B) Hypoxia pretreatment of BM-MSCs increased the ability for tubular formation by HUVECs after 24 h, as compared to the control group; the average length of the tubular formations was calculated. * indicates P<0.05 versus control group. (C,D) H2O2-treated HUVECs exhibited high expression of caspase-3. The CM from hypoxia-pretreated BM-MSCs depressed the expression of caspase-3 and enhanced the expression of Bcl-2. * indicates P<0.05 versus control group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440823&req=5

pone.0126715.g002: Hypoxia pretreatment of BM-MSCs enhanced the capacity for angiogenesis and reduced apoptosis in HUVECs.(A) Hypoxia pretreatment of BM-MSCs enhanced the capacity for migration by HUVECs after 12 h, as compared to the control group; the efficiency of cell migration was detected. * indicates P<0.05 versus control group. (B) Hypoxia pretreatment of BM-MSCs increased the ability for tubular formation by HUVECs after 24 h, as compared to the control group; the average length of the tubular formations was calculated. * indicates P<0.05 versus control group. (C,D) H2O2-treated HUVECs exhibited high expression of caspase-3. The CM from hypoxia-pretreated BM-MSCs depressed the expression of caspase-3 and enhanced the expression of Bcl-2. * indicates P<0.05 versus control group.
Mentions: Furthermore, we observed the effects of hypoxia pretreatment of BM-MSCs on ECs in vitro. Conditioned medium (CM) was prepared from hypoxia-pretreated BM-MSCs that were concentrated 10 times and were used to evaluate the paracrine effect of BM-MSCs on HUVECs as well as the function of CM in vivo (S2 Fig). The results indicated that non-concentrated CM from normoxic BM-MSCs (N-CM) increased the capacity of migration and tube formation on HUVECs as compared to the control group, and this capacity was significantly increased after treatment with CM from hypoxia-pretreated BM-MSCs (H-CM) (Fig 2A and 2B). Increasing the anti-apoptotic function of ECs is important for angiogenesis. Therefore, we investigated the effects of CM on HUVECs apoptosis. The results indicated that H-CM downregulated the expression of caspase-3, which is induced at 4 h by H2O2, and upregulated the expression of Bcl-2 (Fig 2C and 2D). Therefore, H-CM improved the function of HUVECs in vitro.

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