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Hypoxia accelerates vascular repair of endothelial colony-forming cells on ischemic injury via STAT3-BCL3 axis.

Lee SH, Lee JH, Han YS, Ryu JM, Yoon YM, Han HJ - Stem Cell Res Ther (2015)

Bottom Line: Phosphorylations of the JAK2/STAT3 pathway and clonogenic proliferation were enhanced by short-term ECFC culturing under hypoxia, whereas siRNA-targeting of STAT3 significantly reduced these activities.Expression of BCL3, a target molecule of STAT3, was increased in hypo-ECFCs.Hypoxia preconditioning facilitates functional bioactivities of ECFCs by mediating regulation of the STAT3-BCL3 axis.

View Article: PubMed Central - PubMed

Affiliation: Medical Science Research Institute, Soonchunhyang University Seoul Hospital, Seoul, 140-743, Republic of Korea. ykckss1114@nate.com.

ABSTRACT

Introduction: Endothelial colony-forming cells (ECFCs) significantly improve tissue repair by providing regeneration potential within injured cardiovascular tissue. However, ECFC transplantation into ischemic tissue exhibits limited therapeutic efficacy due to poor engraftment in vivo. We established an adequate ex vivo expansion protocol and identified novel modulators that enhance functional bioactivities of ECFCs.

Methods: To augment the regenerative potential of ECFCs, functional bioactivities of hypoxia-preconditioned ECFCs (hypo-ECFCs) were examined.

Results: Phosphorylations of the JAK2/STAT3 pathway and clonogenic proliferation were enhanced by short-term ECFC culturing under hypoxia, whereas siRNA-targeting of STAT3 significantly reduced these activities. Expression of BCL3, a target molecule of STAT3, was increased in hypo-ECFCs. Moreover, siRNA inhibition of BCL3 markedly reduced survival of ECFCs during hypoxic stress in vitro and ischemic stress in vivo. In a hindlimb ischemia model of ischemia, hypo-ECFC transplantation enhanced blood flow ratio, capillary density, transplanted cell proliferation and survival, and angiogenic cytokine secretion at ischemic sites.

Conclusions: Hypoxia preconditioning facilitates functional bioactivities of ECFCs by mediating regulation of the STAT3-BCL3 axis. Thus, a hypoxic preconditioned ex vivo expansion protocol triggers expansion and functional bioactivities of ECFCs via modulation of the hypoxia-induced STAT3-BCL3 axis, suggesting that hypo-ECFCs offer a therapeutic strategy for accelerated neovasculogenesis in ischemic diseases.

No MeSH data available.


Related in: MedlinePlus

Transplanted hypo-ECFCs enhance secretion of vascular endothelial growth factor (VEGF) in ischemic limb muscle via STAT3 signaling. a, b ECFCs, si-STAT3-ECFCs, and scramble siRNA hypo-ECFCs were cultured in normoxic or hypoxic conditions for 12 h, and VEGF levels were determined by using ELISA and western blot analysis. The results are expressed as the mean ± SD. c Western blotting analyses of VEGF conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. d Immunofluorescence staining for VEGF in ischemic tissues conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (n = 7). **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell
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Fig6: Transplanted hypo-ECFCs enhance secretion of vascular endothelial growth factor (VEGF) in ischemic limb muscle via STAT3 signaling. a, b ECFCs, si-STAT3-ECFCs, and scramble siRNA hypo-ECFCs were cultured in normoxic or hypoxic conditions for 12 h, and VEGF levels were determined by using ELISA and western blot analysis. The results are expressed as the mean ± SD. c Western blotting analyses of VEGF conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. d Immunofluorescence staining for VEGF in ischemic tissues conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (n = 7). **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell

Mentions: Transplantation of ECFCs into ischemic tissue enhances the paracrine secretion of angiogenic growth factors such as VEGF. To determine whether VEGF was secreted by hypo-ECFCs, we analyzed the secretion and expression of VEGF by ELISA and western blot analysis. Hypo-ECFCs clearly exhibited higher secretion and expression of VEGF than nor-ECFCs (Fig. 6a, b). To evaluate whether si-STAT3/hypo-ECFCs alter the secretion of VEGF in ischemic tissue, we transplanted hypo-ECFCs, nor-ECFCs, and si-STAT3/hypo-ECFCs into the hindlimb ischemic injury site. Western blot analysis revealed that VEGF expression within this tissue was elevated after hypo-ECFC transplantation, while VEGF expression following transplantation with si-STAT3/hypo-ECFCs or nor-ECFCs was only slightly higher than that of the PBS-treated control (Fig. 6c). Moreover, VEGF expression was detected by immunofluorescence staining in hypo-ECFCs at 3 days after transplantation, whereas minimal VEGF expression was detected in tissue transplanted with si-STAT3/hypo-ECFCs (Fig. 6d). These results suggested that hypo-ECFCs were able to induce the secretion of VEGF, and that the activation of STAT3 was responsible for VEGF induction.Fig. 6


Hypoxia accelerates vascular repair of endothelial colony-forming cells on ischemic injury via STAT3-BCL3 axis.

Lee SH, Lee JH, Han YS, Ryu JM, Yoon YM, Han HJ - Stem Cell Res Ther (2015)

Transplanted hypo-ECFCs enhance secretion of vascular endothelial growth factor (VEGF) in ischemic limb muscle via STAT3 signaling. a, b ECFCs, si-STAT3-ECFCs, and scramble siRNA hypo-ECFCs were cultured in normoxic or hypoxic conditions for 12 h, and VEGF levels were determined by using ELISA and western blot analysis. The results are expressed as the mean ± SD. c Western blotting analyses of VEGF conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. d Immunofluorescence staining for VEGF in ischemic tissues conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (n = 7). **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Transplanted hypo-ECFCs enhance secretion of vascular endothelial growth factor (VEGF) in ischemic limb muscle via STAT3 signaling. a, b ECFCs, si-STAT3-ECFCs, and scramble siRNA hypo-ECFCs were cultured in normoxic or hypoxic conditions for 12 h, and VEGF levels were determined by using ELISA and western blot analysis. The results are expressed as the mean ± SD. c Western blotting analyses of VEGF conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. d Immunofluorescence staining for VEGF in ischemic tissues conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (n = 7). **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell
Mentions: Transplantation of ECFCs into ischemic tissue enhances the paracrine secretion of angiogenic growth factors such as VEGF. To determine whether VEGF was secreted by hypo-ECFCs, we analyzed the secretion and expression of VEGF by ELISA and western blot analysis. Hypo-ECFCs clearly exhibited higher secretion and expression of VEGF than nor-ECFCs (Fig. 6a, b). To evaluate whether si-STAT3/hypo-ECFCs alter the secretion of VEGF in ischemic tissue, we transplanted hypo-ECFCs, nor-ECFCs, and si-STAT3/hypo-ECFCs into the hindlimb ischemic injury site. Western blot analysis revealed that VEGF expression within this tissue was elevated after hypo-ECFC transplantation, while VEGF expression following transplantation with si-STAT3/hypo-ECFCs or nor-ECFCs was only slightly higher than that of the PBS-treated control (Fig. 6c). Moreover, VEGF expression was detected by immunofluorescence staining in hypo-ECFCs at 3 days after transplantation, whereas minimal VEGF expression was detected in tissue transplanted with si-STAT3/hypo-ECFCs (Fig. 6d). These results suggested that hypo-ECFCs were able to induce the secretion of VEGF, and that the activation of STAT3 was responsible for VEGF induction.Fig. 6

Bottom Line: Phosphorylations of the JAK2/STAT3 pathway and clonogenic proliferation were enhanced by short-term ECFC culturing under hypoxia, whereas siRNA-targeting of STAT3 significantly reduced these activities.Expression of BCL3, a target molecule of STAT3, was increased in hypo-ECFCs.Hypoxia preconditioning facilitates functional bioactivities of ECFCs by mediating regulation of the STAT3-BCL3 axis.

View Article: PubMed Central - PubMed

Affiliation: Medical Science Research Institute, Soonchunhyang University Seoul Hospital, Seoul, 140-743, Republic of Korea. ykckss1114@nate.com.

ABSTRACT

Introduction: Endothelial colony-forming cells (ECFCs) significantly improve tissue repair by providing regeneration potential within injured cardiovascular tissue. However, ECFC transplantation into ischemic tissue exhibits limited therapeutic efficacy due to poor engraftment in vivo. We established an adequate ex vivo expansion protocol and identified novel modulators that enhance functional bioactivities of ECFCs.

Methods: To augment the regenerative potential of ECFCs, functional bioactivities of hypoxia-preconditioned ECFCs (hypo-ECFCs) were examined.

Results: Phosphorylations of the JAK2/STAT3 pathway and clonogenic proliferation were enhanced by short-term ECFC culturing under hypoxia, whereas siRNA-targeting of STAT3 significantly reduced these activities. Expression of BCL3, a target molecule of STAT3, was increased in hypo-ECFCs. Moreover, siRNA inhibition of BCL3 markedly reduced survival of ECFCs during hypoxic stress in vitro and ischemic stress in vivo. In a hindlimb ischemia model of ischemia, hypo-ECFC transplantation enhanced blood flow ratio, capillary density, transplanted cell proliferation and survival, and angiogenic cytokine secretion at ischemic sites.

Conclusions: Hypoxia preconditioning facilitates functional bioactivities of ECFCs by mediating regulation of the STAT3-BCL3 axis. Thus, a hypoxic preconditioned ex vivo expansion protocol triggers expansion and functional bioactivities of ECFCs via modulation of the hypoxia-induced STAT3-BCL3 axis, suggesting that hypo-ECFCs offer a therapeutic strategy for accelerated neovasculogenesis in ischemic diseases.

No MeSH data available.


Related in: MedlinePlus