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Cited by 103Mesenchymal stem cell researchAngiogenesis and VEGF in CancerTissue Engineering and Regenerative Medicine

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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

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Liu, Jiejie, Hao, Haojie, Xia, Lei, Ti, Dongdong, Huang, Hong, Dong, Liang, Tong, Chuan, Hou, Qian, Zhao, Yali, Liu, Huiling, Fu, Xiaobing, Han, Weidong·2015·PLOS ONE
DOI 10.1371/journal.pone.0126715PMC PMC4440823
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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.

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Fig 1 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,.
Fig 1 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,.CC BY · PubMed Central
Fig 2 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.
Fig 2 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.CC BY · PubMed Central
Fig 3 Hypoxia pretreatment of BM-MSCs modulates VEGF/AKT signaling in HUVECs. (A) The expression of VEGF-1α was suppressed with lentiviral-mediated siRNA-VEGF-1α (siVEGF). (B) CM generated from hypoxia pretreated BM-MSCs treated with siVEGF downregulated the expression of pAKT and VEGFR in HUVECs, as determined by western blot. (C) The capacity of HUVECs to form tubes was inhibited by CM from VEGF-1α-silenced BM-MSCs.
Fig 3 Hypoxia pretreatment of BM-MSCs modulates VEGF/AKT signaling in HUVECs. (A) The expression of VEGF-1α was suppressed with lentiviral-mediated siRNA-VEGF-1α (siVEGF). (B) CM generated from hypoxia pretreated BM-MSCs treated with siVEGF downregulated the expression of pAKT and VEGFR in HUVECs, as determined by western blot. (C) The capacity of HUVECs to form tubes was inhibited by CM from VEGF-1α-silenced BM-MSCs.CC BY · PubMed Central
Fig 4 Hypoxia pretreatment of BM-MSCs promoted angiogenesis in ischemic muscle, as evaluated by angiography. (A) We presented the block diagram of study for BM-MSCs transplanted to treat DLLI. (B) The model of DLLI was developed and confirmed by angiography. (C) Hypoxia pretreatment of BM-MSCs locally transplanted to ischemic muscle markedly enhanced the microvascular density, compared to the control group. (D) The average length of the capillaries was detected. n = 4. * indicates P<0.05 versus the 7 d control group. (E) Perfusion images of lower limb at 14 days postoperatively. The color scale of all images was analyzed by setting the lowest perfusion value to 0 and the highest perfusion value to 300. (F)Perfusion in the normoxia and control groups was lower than that in the hypoxia group (* indicates P<0.05).
Fig 4 Hypoxia pretreatment of BM-MSCs promoted angiogenesis in ischemic muscle, as evaluated by angiography. (A) We presented the block diagram of study for BM-MSCs transplanted to treat DLLI. (B) The model of DLLI was developed and confirmed by angiography. (C) Hypoxia pretreatment of BM-MSCs locally transplanted to ischemic muscle markedly enhanced the microvascular density, compared to the control group. (D) The average length of the capillaries was detected. n = 4. * indicates P<0.05 versus the 7 d control group. (E) Perfusion images of lower limb at 14 days postoperatively. The color scale of all images was analyzed by setting the lowest perfusion value to 0 and the highest perfusion value to 300. (F)Perfusion in the normoxia and control groups was lower than that in the hypoxia group (* indicates P<0.05).CC BY · PubMed Central
Fig 5 Hypoxia pretreatment of BM-MSCs promoted angiogenesis in DLLI muscle, as assessed by immunohistochemical staining. (A) Histological analysis of muscle tissues was performed by immunostaining. The specimens were stained with H&E (hematoxylin-eosin). Hypoxia pretreatment of BM-MSCs accelerated the repair of muscle fibers at day 7. At day 14, the hypoxia pretreatment BM-MSCs group exhibited a de novo rebound in the microvasculature. (B) Immunohistochemical staining with a specific antibody recognizing the endothelial marker CD31 revealed the presence of more mature vessels (muscle), predominantly in the hypoxia pretreatment BM-MSCs group rather than in the control group, and (C) the number of vessels was measured.*, ** indicate P<0.05, 0.01 versus control group, respectively.
Fig 5 Hypoxia pretreatment of BM-MSCs promoted angiogenesis in DLLI muscle, as assessed by immunohistochemical staining. (A) Histological analysis of muscle tissues was performed by immunostaining. The specimens were stained with H&E (hematoxylin-eosin). Hypoxia pretreatment of BM-MSCs accelerated the repair of muscle fibers at day 7. At day 14, the hypoxia pretreatment BM-MSCs group exhibited a de novo rebound in the microvasculature. (B) Immunohistochemical staining with a specific antibody recognizing the endothelial marker CD31 revealed the presence of more mature vessels (muscle), predominantly in the hypoxia pretreatment BM-MSCs group rather than in the control group, and (C) the number of vessels was measured.*, ** indicate P<0.05, 0.01 versus control group, respectively.CC BY · PubMed Central
Fig 6 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.
Fig 6 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.CC BY · PubMed Central
Fig 7 Hypoxia pretreatment of BM-MSCs improved the function of ECs in vivo. (A, B) Immunohistochemical staining with specific antibodies against the endothelial marker vWF and cell proliferation marker Ki67 revealed more double-positive cells in the hypoxia pretreated BM-MSC group than in the control group. * indicates P<0.05 versus control group. (C, D) Immunohistochemical staining with a specific antibody recognizing the endothelial marker vWF and cell apoptosis marker TUNEL revealed that hypoxia pretreatment of BM-MSCs decreased the number of double-positive cells. * indicates P<0.05 versus control group. (E,F) Hypoxia pretreatment of BM-MSCs upregulated the expression of Bcl-2 in muscle and reduced the expression of caspase-3. * indicates P<0.05 versus control group. * indicates P<0.05 versus control group.
Fig 7 Hypoxia pretreatment of BM-MSCs improved the function of ECs in vivo. (A, B) Immunohistochemical staining with specific antibodies against the endothelial marker vWF and cell proliferation marker Ki67 revealed more double-positive cells in the hypoxia pretreated BM-MSC group than in the control group. * indicates P<0.05 versus control group. (C, D) Immunohistochemical staining with a specific antibody recognizing the endothelial marker vWF and cell apoptosis marker TUNEL revealed that hypoxia pretreatment of BM-MSCs decreased the number of double-positive cells. * indicates P<0.05 versus control group. (E,F) Hypoxia pretreatment of BM-MSCs upregulated the expression of Bcl-2 in muscle and reduced the expression of caspase-3. * indicates P<0.05 versus control group. * indicates P<0.05 versus control group.CC BY · PubMed Central
Fig 8 Hypoxia-pretreated BM-MSCs significantly increased the survival of the BM-MSCs in ischemic muscle, markedly increased angiogenesis and improved the proliferation of endothelial cells in ischemic muscle.
Fig 8 Hypoxia-pretreated BM-MSCs significantly increased the survival of the BM-MSCs in ischemic muscle, markedly increased angiogenesis and improved the proliferation of endothelial cells in ischemic muscle.CC BY · PubMed Central

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Metadata

Authors
Liu, J., Hao, H., Xia, L., Ti, D., Huang, H., Dong, L., Tong, C., Hou, Q., Zhao, Y., Liu, H., Fu, X., & Han, W.
Affiliations
Chinese PLA General Hospital (CN)
Corresponding author
  • Fu, Xiaobing
  • Han, Weidong
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-05-21
Volume
10
Issue
5
Pages
e0126715
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0126715
PMC
PMC4440823
OpenAlex
W1506316383
Cited by
101
References
37
Citations by year
2026: 4 · 2025: 4 · 2024: 12 · 2023: 13 · 2022: 11 · 2021: 7 · 2020: 14 · 2019: 6 · 2018: 10 · 2017: 7 · 2016: 10 · 2015: 3
FWCI
5.60
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
National Natural Science Foundation of China (2012CB518105, 81121004, 81230041, 2013AA020105, 2012AA020502, 2012CB518103)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Keywords
bone marrow mesenchymal stem cells, angiogenesis, endothelial dysfunction, revascularization, HIF-1α, MMP-9, Bcl-2, cell survival after transplantation, Akt signaling, angiogenic factors
MeSH terms
Angiography, Animals, Hypoxia, Cell Survival, Cells, Cultured, Diabetes Mellitus, Experimental, Disease Models, Animal, Humans, Ischemia, Male, Neovascularization, Pathologic, Ultrasonography, Signal Transduction, Apoptosis, Lower Extremity, Vascular Endothelial Growth Factors, Endothelial Cells, Angiogenesis Inducing Agents, Mesenchymal Stem Cell Transplantation, Proto-Oncogene Proteins c-akt, Rats, Mesenchymal Stem Cells, Human Umbilical Vein Endothelial Cells

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