Limits...
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

Activation of the JAK2/STAT3 signaling pathway in hypo-ECFCs and the regulation of proliferative, clonogenic potential. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for 0–24 h, and JAK2, p-JAK2, STAT3, and p-STAT3 were detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA and scramble siRNA for 48 h prior to hypoxia exposure for 24 h. p-STAT3 was detected by western blot analysis. c Representative photomicrograph of the cell clusters derived from single nor-ECFCs, hypo-ECFCs, STAT3-siRNA-transfected hypo-ECFCs (si-STAT3/hypo-ECFCs), and scramble siRNA-transfected hypo-ECFCs, as well as d a graph of the number of these cells per colony. e Images of hematoxylin-stained plates with representative nor-ECFC, hypo-ECFC, si-STAT3/hypo-ECFC, and scramble siRNA-transfected hypo-ECFCs colonies, as well as f graphs of the number of these colonies per 96-well plate. The example shown is representative of four independent experiments. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4522108&req=5

Fig2: Activation of the JAK2/STAT3 signaling pathway in hypo-ECFCs and the regulation of proliferative, clonogenic potential. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for 0–24 h, and JAK2, p-JAK2, STAT3, and p-STAT3 were detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA and scramble siRNA for 48 h prior to hypoxia exposure for 24 h. p-STAT3 was detected by western blot analysis. c Representative photomicrograph of the cell clusters derived from single nor-ECFCs, hypo-ECFCs, STAT3-siRNA-transfected hypo-ECFCs (si-STAT3/hypo-ECFCs), and scramble siRNA-transfected hypo-ECFCs, as well as d a graph of the number of these cells per colony. e Images of hematoxylin-stained plates with representative nor-ECFC, hypo-ECFC, si-STAT3/hypo-ECFC, and scramble siRNA-transfected hypo-ECFCs colonies, as well as f graphs of the number of these colonies per 96-well plate. The example shown is representative of four independent experiments. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC

Mentions: To assess the involvement of JAK2/STAT3 in hypoxic preconditioning of the proliferative and clonogenic potential of ECFCs, hypoxia-induced phosphorylation of JAK2 and STAT3 was examined by western blot. Hypoxia increased JAK2 and STAT3 phosphorylation in a time-dependent manner (Fig. 2a). We then examined whether STAT3 plays a positive role in the regulation of the proliferative and clonogenic potential in ECFC. The hypoxia-induced increase in STAT3 phosphorylation was attenuated by STAT3-specific siRNAs (Fig. 2b). Correspondingly, siRNA targeting of STAT3 in hypoxic cells decreased single-cell division as well as the number of colonies (Fig. 2c–f). This suggests that hypoxia increases proliferative and clonogenic potential through the activation of STAT3.Fig. 2


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)

Activation of the JAK2/STAT3 signaling pathway in hypo-ECFCs and the regulation of proliferative, clonogenic potential. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for 0–24 h, and JAK2, p-JAK2, STAT3, and p-STAT3 were detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA and scramble siRNA for 48 h prior to hypoxia exposure for 24 h. p-STAT3 was detected by western blot analysis. c Representative photomicrograph of the cell clusters derived from single nor-ECFCs, hypo-ECFCs, STAT3-siRNA-transfected hypo-ECFCs (si-STAT3/hypo-ECFCs), and scramble siRNA-transfected hypo-ECFCs, as well as d a graph of the number of these cells per colony. e Images of hematoxylin-stained plates with representative nor-ECFC, hypo-ECFC, si-STAT3/hypo-ECFC, and scramble siRNA-transfected hypo-ECFCs colonies, as well as f graphs of the number of these colonies per 96-well plate. The example shown is representative of four independent experiments. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522108&req=5

Fig2: Activation of the JAK2/STAT3 signaling pathway in hypo-ECFCs and the regulation of proliferative, clonogenic potential. a Endothelial colony-forming cells (ECFCs) were exposed to hypoxia for 0–24 h, and JAK2, p-JAK2, STAT3, and p-STAT3 were detected by western blot analysis. b ECFCs were transfected with STAT3-siRNA and scramble siRNA for 48 h prior to hypoxia exposure for 24 h. p-STAT3 was detected by western blot analysis. c Representative photomicrograph of the cell clusters derived from single nor-ECFCs, hypo-ECFCs, STAT3-siRNA-transfected hypo-ECFCs (si-STAT3/hypo-ECFCs), and scramble siRNA-transfected hypo-ECFCs, as well as d a graph of the number of these cells per colony. e Images of hematoxylin-stained plates with representative nor-ECFC, hypo-ECFC, si-STAT3/hypo-ECFC, and scramble siRNA-transfected hypo-ECFCs colonies, as well as f graphs of the number of these colonies per 96-well plate. The example shown is representative of four independent experiments. **P < 0.01 vs. nor-ECFCs; ##P < 0.01 vs. hypo-ECFCs; $$P < 0.01 vs. si-STAT3/hypo-ECFC
Mentions: To assess the involvement of JAK2/STAT3 in hypoxic preconditioning of the proliferative and clonogenic potential of ECFCs, hypoxia-induced phosphorylation of JAK2 and STAT3 was examined by western blot. Hypoxia increased JAK2 and STAT3 phosphorylation in a time-dependent manner (Fig. 2a). We then examined whether STAT3 plays a positive role in the regulation of the proliferative and clonogenic potential in ECFC. The hypoxia-induced increase in STAT3 phosphorylation was attenuated by STAT3-specific siRNAs (Fig. 2b). Correspondingly, siRNA targeting of STAT3 in hypoxic cells decreased single-cell division as well as the number of colonies (Fig. 2c–f). This suggests that hypoxia increases proliferative and clonogenic potential through the activation of STAT3.Fig. 2

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