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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

The characterization of rat BM-MSCs cultured under hypoxic conditions.(A) The expression of VEGF-1α, HIF-1α, ANG-1, bFGF, and MMP-9 were significantly increased in BM-MSCs receiving hypoxia pretreatment for 24 h compared to normoxic controls. At 48 h or 72 h, hypoxia pretreatment at 5% O2 continuously increased the expression of these genes. (B) The expression of the VEGF-1a protein was significantly increased in BM-MSCs after hypoxia pretreatment. (C) The expression of the anti-apoptosis protein Bcl-2 was enhanced by western blot. * indicates P<0.05 versus the normoxia group, and (D) the apoptosis of BM-MSCs was analyzed by flow cytometry for cells cultured with 5% O2. At 48 h, no changes were observed in vitro,.
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pone.0126715.g001: The characterization of rat BM-MSCs cultured under hypoxic conditions.(A) The expression of VEGF-1α, HIF-1α, ANG-1, bFGF, and MMP-9 were significantly increased in BM-MSCs receiving hypoxia pretreatment for 24 h compared to normoxic controls. At 48 h or 72 h, hypoxia pretreatment at 5% O2 continuously increased the expression of these genes. (B) The expression of the VEGF-1a protein was significantly increased in BM-MSCs after hypoxia pretreatment. (C) The expression of the anti-apoptosis protein Bcl-2 was enhanced by western blot. * indicates P<0.05 versus the normoxia group, and (D) the apoptosis of BM-MSCs was analyzed by flow cytometry for cells cultured with 5% O2. At 48 h, no changes were observed in vitro,.

Mentions: To characterize rat BM-MSCs cultured under hypoxic conditions, BM-MSCs were isolated and cultured with different oxygen concentrations for different times. The results demonstrated that the expression of VEGF-1a, HIF-1a, HGF, bFGF, MMP-9, and PDGF was enhanced in BM-MSCs cultured under hypoxia (2%, 5%, or 7%), as determined by real time RT-PCR. However, we found that the expression of those genes was transient, with gene expression most markedly increased in BM-MSCs cultured in 5% O2 for 48 h. In particular, the expression of VEGF-1a and HIF was highly upregulated, compared to the other genes (Fig 1A). Western blotting showed that the expression of VEGF-1a was consistent with the gene expression results (Fig 1B). For this reason and because VEGF-1a and HIF play important roles in promoting angiogenesis, the BM-MSC pretreatment condition of 5% O2 for 48 h was chosen for this study. We further observed the transient effect of pretreatment with 5% O2 for 48 h on BM-MSC apoptosis by flow cytometry. The results indicated that the percentage of positive (Annexin V+, PI+) cells after hypoxia pretreatment was similar to those cultured under normoxic conditions (Fig 1D). Moreover, hypoxic pretreatment had no effect on the cell phenotype or adipogenic and osteogenic differentiation abilities. We further examined the phenotypic changes of BM-MSCs under 5% oxygen pretreatment and found that the phenotype of BM-MSCs showed no obvious change after hypoxic preconditioning (S1 Fig). Western blotting indicated that pretreated BM-MSCs exhibited enhanced expression of the anti-apoptotic protein Bcl-2 (1:500, Cell Signaling), while the expression of apoptotic protein caspase-3 (1:500, Cell Signaling) was not altered (Fig 1C).


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)

The characterization of rat BM-MSCs cultured under hypoxic conditions.(A) The expression of VEGF-1α, HIF-1α, ANG-1, bFGF, and MMP-9 were significantly increased in BM-MSCs receiving hypoxia pretreatment for 24 h compared to normoxic controls. At 48 h or 72 h, hypoxia pretreatment at 5% O2 continuously increased the expression of these genes. (B) The expression of the VEGF-1a protein was significantly increased in BM-MSCs after hypoxia pretreatment. (C) The expression of the anti-apoptosis protein Bcl-2 was enhanced by western blot. * indicates P<0.05 versus the normoxia group, and (D) the apoptosis of BM-MSCs was analyzed by flow cytometry for cells cultured with 5% O2. At 48 h, no changes were observed in vitro,.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0126715.g001: The characterization of rat BM-MSCs cultured under hypoxic conditions.(A) The expression of VEGF-1α, HIF-1α, ANG-1, bFGF, and MMP-9 were significantly increased in BM-MSCs receiving hypoxia pretreatment for 24 h compared to normoxic controls. At 48 h or 72 h, hypoxia pretreatment at 5% O2 continuously increased the expression of these genes. (B) The expression of the VEGF-1a protein was significantly increased in BM-MSCs after hypoxia pretreatment. (C) The expression of the anti-apoptosis protein Bcl-2 was enhanced by western blot. * indicates P<0.05 versus the normoxia group, and (D) the apoptosis of BM-MSCs was analyzed by flow cytometry for cells cultured with 5% O2. At 48 h, no changes were observed in vitro,.
Mentions: To characterize rat BM-MSCs cultured under hypoxic conditions, BM-MSCs were isolated and cultured with different oxygen concentrations for different times. The results demonstrated that the expression of VEGF-1a, HIF-1a, HGF, bFGF, MMP-9, and PDGF was enhanced in BM-MSCs cultured under hypoxia (2%, 5%, or 7%), as determined by real time RT-PCR. However, we found that the expression of those genes was transient, with gene expression most markedly increased in BM-MSCs cultured in 5% O2 for 48 h. In particular, the expression of VEGF-1a and HIF was highly upregulated, compared to the other genes (Fig 1A). Western blotting showed that the expression of VEGF-1a was consistent with the gene expression results (Fig 1B). For this reason and because VEGF-1a and HIF play important roles in promoting angiogenesis, the BM-MSC pretreatment condition of 5% O2 for 48 h was chosen for this study. We further observed the transient effect of pretreatment with 5% O2 for 48 h on BM-MSC apoptosis by flow cytometry. The results indicated that the percentage of positive (Annexin V+, PI+) cells after hypoxia pretreatment was similar to those cultured under normoxic conditions (Fig 1D). Moreover, hypoxic pretreatment had no effect on the cell phenotype or adipogenic and osteogenic differentiation abilities. We further examined the phenotypic changes of BM-MSCs under 5% oxygen pretreatment and found that the phenotype of BM-MSCs showed no obvious change after hypoxic preconditioning (S1 Fig). Western blotting indicated that pretreated BM-MSCs exhibited enhanced expression of the anti-apoptotic protein Bcl-2 (1:500, Cell Signaling), while the expression of apoptotic protein caspase-3 (1:500, Cell Signaling) was not altered (Fig 1C).

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