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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

STAT3-mediated transplanted hypo-ECFC proliferation in ischemic tissues. a Immunofluorescence staining for proliferating cell nuclear antigen (PCNA, green) in ischemic hindlimb after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (scale bar: 100 μm). b Co-immunofluorescence staining to detect Ki-67 (a proliferation marker, red) and nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining (scale bar: 50 μm). c Bar graph shows the results of number of PCNA+ cells 3 days after transplantation. d Quantitative analysis of Ki-67/HNA/DAPI triple-positive cells 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell, PBS phosphate-buffered saline
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Fig4: STAT3-mediated transplanted hypo-ECFC proliferation in ischemic tissues. a Immunofluorescence staining for proliferating cell nuclear antigen (PCNA, green) in ischemic hindlimb after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (scale bar: 100 μm). b Co-immunofluorescence staining to detect Ki-67 (a proliferation marker, red) and nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining (scale bar: 50 μm). c Bar graph shows the results of number of PCNA+ cells 3 days after transplantation. d Quantitative analysis of Ki-67/HNA/DAPI triple-positive cells 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell, PBS phosphate-buffered saline

Mentions: In the previous in vitro experiments, we demonstrated that the culture of ECFCs in hypoxic conditions activated the STAT3 signaling pathway and increased their proliferative potential. We hypothesized that hypoxic-preconditioned culturing of ECFCs (hypo-ECFCs) would be beneficial for repairing the damaged tissue in the hindlimb ischemia injury model by providing cells with a better proliferative rate to the site of ischemic injury. To evaluate the in vivo regulation of the STAT3 pathway, we transplanted nor-ECFCs and hypo-ECFCs into nude mice with hindlimb ischemia. After 3 days, ischemic tissues transplanted with hypo-ECFCs exhibited significantly higher levels of STAT3 activation than tissues transplanted with nor-ECFCs (Fig. 3a). Next, the distribution of STAT3 in hypo-ECFCs was evaluated. Hypo-ECFCs exhibited an increase in the number of phospho-STAT3/HNA/DAPI-positive cells in the ECFC population (Fig. 3b). Accordingly, more proliferating cell nuclear antigen (PCNA)-positive cells were found in tissue transplanted with hypo-ECFCs than that transplanted with nor-ECFCs (Fig. 4a, c). HNA and proliferating cell marker (Ki-67)-double positive cells were more abundant at 3 days in tissue receiving hypo-ECFCs than that receiving nor-ECFCs (Fig. 4b, d). However, STAT3-specific siRNA-transfected hypo-ECFCs (si-STAT3/hypo-ECFCs) displayed poor proliferation in ischemic tissue.Fig. 3


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)

STAT3-mediated transplanted hypo-ECFC proliferation in ischemic tissues. a Immunofluorescence staining for proliferating cell nuclear antigen (PCNA, green) in ischemic hindlimb after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (scale bar: 100 μm). b Co-immunofluorescence staining to detect Ki-67 (a proliferation marker, red) and nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining (scale bar: 50 μm). c Bar graph shows the results of number of PCNA+ cells 3 days after transplantation. d Quantitative analysis of Ki-67/HNA/DAPI triple-positive cells 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell, PBS phosphate-buffered saline
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Fig4: STAT3-mediated transplanted hypo-ECFC proliferation in ischemic tissues. a Immunofluorescence staining for proliferating cell nuclear antigen (PCNA, green) in ischemic hindlimb after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (scale bar: 100 μm). b Co-immunofluorescence staining to detect Ki-67 (a proliferation marker, red) and nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining (scale bar: 50 μm). c Bar graph shows the results of number of PCNA+ cells 3 days after transplantation. d Quantitative analysis of Ki-67/HNA/DAPI triple-positive cells 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-STAT3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC. ECFC endothelial colony-forming cell, PBS phosphate-buffered saline
Mentions: In the previous in vitro experiments, we demonstrated that the culture of ECFCs in hypoxic conditions activated the STAT3 signaling pathway and increased their proliferative potential. We hypothesized that hypoxic-preconditioned culturing of ECFCs (hypo-ECFCs) would be beneficial for repairing the damaged tissue in the hindlimb ischemia injury model by providing cells with a better proliferative rate to the site of ischemic injury. To evaluate the in vivo regulation of the STAT3 pathway, we transplanted nor-ECFCs and hypo-ECFCs into nude mice with hindlimb ischemia. After 3 days, ischemic tissues transplanted with hypo-ECFCs exhibited significantly higher levels of STAT3 activation than tissues transplanted with nor-ECFCs (Fig. 3a). Next, the distribution of STAT3 in hypo-ECFCs was evaluated. Hypo-ECFCs exhibited an increase in the number of phospho-STAT3/HNA/DAPI-positive cells in the ECFC population (Fig. 3b). Accordingly, more proliferating cell nuclear antigen (PCNA)-positive cells were found in tissue transplanted with hypo-ECFCs than that transplanted with nor-ECFCs (Fig. 4a, c). HNA and proliferating cell marker (Ki-67)-double positive cells were more abundant at 3 days in tissue receiving hypo-ECFCs than that receiving nor-ECFCs (Fig. 4b, d). However, STAT3-specific siRNA-transfected hypo-ECFCs (si-STAT3/hypo-ECFCs) displayed poor proliferation in ischemic tissue.Fig. 3

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