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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

Modulation of the STAT3-BCL3 axis enhances survival induced by ischemic stress. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for various times (0–24 h). BCL3 expression was detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA for 48 h prior to exposure to hypoxic conditions for 24 h. BCL3 was detected by western blot analysis. c ECFCs were transfected with BCL3-siRNA for 48 h prior to exposure to hypoxic conditions for 96 h. Cleaved caspase-3 was detected by western blot analysis. d ECFCs were transfected with BCL3-siRNA for 48 h prior to treatment with H2O2 (10−3 M) for 8 h followed by western blot analysis for cleaved caspase-3. e Co-immunofluorescence staining was used to detect apoptosis (caspase-3, an apoptosis marker, red) and nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining. Arrows indicate caspase-3+/HNA+/DAPI+ cells (scale bar: 50 μm). f Quantitative analysis of caspase-3/HNA/DAPI triple-positive cells conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC; n = 10
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Fig5: Modulation of the STAT3-BCL3 axis enhances survival induced by ischemic stress. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for various times (0–24 h). BCL3 expression was detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA for 48 h prior to exposure to hypoxic conditions for 24 h. BCL3 was detected by western blot analysis. c ECFCs were transfected with BCL3-siRNA for 48 h prior to exposure to hypoxic conditions for 96 h. Cleaved caspase-3 was detected by western blot analysis. d ECFCs were transfected with BCL3-siRNA for 48 h prior to treatment with H2O2 (10−3 M) for 8 h followed by western blot analysis for cleaved caspase-3. e Co-immunofluorescence staining was used to detect apoptosis (caspase-3, an apoptosis marker, red) and nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining. Arrows indicate caspase-3+/HNA+/DAPI+ cells (scale bar: 50 μm). f Quantitative analysis of caspase-3/HNA/DAPI triple-positive cells conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC; n = 10

Mentions: Next, we determined whether hypoxia-induced activation of STAT3 led to the activation of BCL3, a known target of STAT3 [28], subsequently promoting cell survival [17]. Normoxic cells were found to express a very low level of BCL3, whereas hypoxic cells exhibited elevated BCL3 expression (Fig. 5a). We examined whether STAT3 was involved in the regulation of BCL3 expression. As expected, the hypoxia-induced increase in BCL3 expression was attenuated by STAT3-specific siRNAs (Fig. 5b). Additionally, siRNA-targeting BCL3 in long-term hypoxic conditions (96 h) in the presence of a high concentration of H2O2 (10−3 M) increased cleaved caspase-3 levels, a marker for apoptosis, relative to hypoxia alone (Fig. 5c, d). Furthermore, immunofluorescence staining for caspase-3 and HNA in ischemic muscle at 3 days after transplantation revealed that apoptotic ECFCs were significantly reduced after transplantation with hypo-ECFCs compared to after transplantation with nor-ECFCs, but BCL3-specific siRNA-transfected hypo-ECFCs (si-BCL3/hypo-ECFCs) displayed poor survival in ischemic tissue (Fig. 5e, f). These data suggest that STAT3 increases cell survival and prevents apoptosis via the induction of BCL3.Fig. 5


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)

Modulation of the STAT3-BCL3 axis enhances survival induced by ischemic stress. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for various times (0–24 h). BCL3 expression was detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA for 48 h prior to exposure to hypoxic conditions for 24 h. BCL3 was detected by western blot analysis. c ECFCs were transfected with BCL3-siRNA for 48 h prior to exposure to hypoxic conditions for 96 h. Cleaved caspase-3 was detected by western blot analysis. d ECFCs were transfected with BCL3-siRNA for 48 h prior to treatment with H2O2 (10−3 M) for 8 h followed by western blot analysis for cleaved caspase-3. e Co-immunofluorescence staining was used to detect apoptosis (caspase-3, an apoptosis marker, red) and nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining. Arrows indicate caspase-3+/HNA+/DAPI+ cells (scale bar: 50 μm). f Quantitative analysis of caspase-3/HNA/DAPI triple-positive cells conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC; n = 10
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Fig5: Modulation of the STAT3-BCL3 axis enhances survival induced by ischemic stress. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for various times (0–24 h). BCL3 expression was detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA for 48 h prior to exposure to hypoxic conditions for 24 h. BCL3 was detected by western blot analysis. c ECFCs were transfected with BCL3-siRNA for 48 h prior to exposure to hypoxic conditions for 96 h. Cleaved caspase-3 was detected by western blot analysis. d ECFCs were transfected with BCL3-siRNA for 48 h prior to treatment with H2O2 (10−3 M) for 8 h followed by western blot analysis for cleaved caspase-3. e Co-immunofluorescence staining was used to detect apoptosis (caspase-3, an apoptosis marker, red) and nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs (human nuclear antigen (HNA)-positive cells, green). DAPI (blue) was used for nuclear staining. Arrows indicate caspase-3+/HNA+/DAPI+ cells (scale bar: 50 μm). f Quantitative analysis of caspase-3/HNA/DAPI triple-positive cells conducted 3 days after transplantation of nor-ECFCs, hypo-ECFCs, si-BCL3/hypo-ECFCs, or scramble siRNA/hypo-ECFCs. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC; n = 10
Mentions: Next, we determined whether hypoxia-induced activation of STAT3 led to the activation of BCL3, a known target of STAT3 [28], subsequently promoting cell survival [17]. Normoxic cells were found to express a very low level of BCL3, whereas hypoxic cells exhibited elevated BCL3 expression (Fig. 5a). We examined whether STAT3 was involved in the regulation of BCL3 expression. As expected, the hypoxia-induced increase in BCL3 expression was attenuated by STAT3-specific siRNAs (Fig. 5b). Additionally, siRNA-targeting BCL3 in long-term hypoxic conditions (96 h) in the presence of a high concentration of H2O2 (10−3 M) increased cleaved caspase-3 levels, a marker for apoptosis, relative to hypoxia alone (Fig. 5c, d). Furthermore, immunofluorescence staining for caspase-3 and HNA in ischemic muscle at 3 days after transplantation revealed that apoptotic ECFCs were significantly reduced after transplantation with hypo-ECFCs compared to after transplantation with nor-ECFCs, but BCL3-specific siRNA-transfected hypo-ECFCs (si-BCL3/hypo-ECFCs) displayed poor survival in ischemic tissue (Fig. 5e, f). These data suggest that STAT3 increases cell survival and prevents apoptosis via the induction of BCL3.Fig. 5

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