Korean J Physiol Pharmacol.  2018 Mar;22(2):203-213. 10.4196/kjpp.2018.22.2.203.

Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation

• Kim DY
• Jung SY
• Kim YJ
• Kang S
• Park JH
• Ji ST
• Jang WB
• Lamichane
• Lamichane
• Chae YC
• Lee D
• Chung JS
• Kwon SM

• KMID: 2410110
• DOI: http://doi.org/10.4196/kjpp.2018.22.2.203

Abstract

Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis.

Keyword

Angiogenesis; DRP1; Endothelial progenitor cells; Hypoxia; Mitochondrial dynamics

MeSH Terms

Anoxia
Cell Movement
Endothelial Cells
Endothelial Progenitor Cells*
Mitochondrial Dynamics*
Oxygen
RNA, Small Interfering
Oxygen
RNA, Small Interfering

Figure

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  Fig. 1 Hypoxia enhances EPC bioactivities. (A) Human EPCs were incubated under 1% O2 hypoxic or 20% O2 for 24 h. The migration assay was performed with a Boyden chamber. Cell migration was visualized by microscopy (magnification 200×). (B) Quantification of the number of migrating cells. Values represent mean±SEM. Experiments were performed in triplicates. **p<0.01. (C) Invasion assays was conducted with or without hypoxia in EPCs. (D) Quantification of invading cells. Values represent mean±SEM. Experiments were performed in triplicates. *p<0.05. (E) Representative images of hypoxia-induced tube formation in EPCs. EPCs were cultured on Matrigel GFR under normal or hypoxic conditions for 18 h. (F) Quantification of total tube length was performed using the Image J software. Experiments were conducted in triplicates. **p<0.01, as compared with that of control. (G) EPCs surface marker expression analyzed by flow cytometry.

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  Fig. 2 Hypoxia-induced mitochondrial fission in EPCs. (A) Human EPCs were cultured under hypoxia or normoxia for 24 h, and mitochondrial fission was observed using confocal microscopy. TOM20 was used as a mitochondrial marker, and nuclei were stained with DAPI. The white square represents magnification, as shown in the bottom panel. Scale bar=10 µm. (B) Hypoxia time-dependent mitochondrial dynamics-related gene expression was determined via immunoblot. EPCs were incubated under 1% O2 hypoxia or normoxia for the indicated time, and cells were harvested. Total DRP1, pDRP1 (S616), pDRP1 (S637), were used as mitochondrial fission markers, and OPA1, MFN1 were used as the markers of mitochondrial fusion, HIF-1A was used as a hypoxic marker, and β-actin was used for loading control. (C) Quantification of pDRP1 (S637) expression was attenuated during hypoxia. Three independent experiment were performed to statistical analysis. *p<0.05.

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  Fig. 3 DRP1-dependent mitochondrial fission in EPCs. (A, B) EPCs were treated with Mdivi-1 for 24 h with serially diluted Mdivi-1 (0–100 µM) in full (A) or starvation (B) media. Cell viability was analyzed and quantified using the CCK-8 assay. Values were normalized to negative control. All experiments were performed in at least triplicates. **p<0.01, ***p<0.001. (C) Cells were treated with 12.5 µM Mdivi-1, and cultured at 1% O2 for 24 h. Mitochondrial morphology was observed using confocal microscopy. White square indicates magnification, as shown in bottom panels. Scale bar=10 µm. TOM20 was used for mitochondrial staining, and DAPI was used for nuclear staining.

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  Fig. 4 Mdivi-1 inhibits EPCs bioactivities under hypoxic condition. (A) EPCs were pre-treated with 12.5 µM Mdivi-1 for 24 h. Cells were seeded into the upper chamber with or without hypoxia for an additional 24 h. (B) Mdivi-1 pre-treated and untreated cells were seeded into Matrigel-coated transwell, and were cultured under normoxia or hypoxia for 24 h. Representative image from three independent experiments is shown. (C, D) Migrating (C) and invading cells (D) were quantified using the Image J software. All experiments were performed in at least triplicates. *p<0.05, **p<0.01. (E) EPCs were pretreated with 12.5 µM Mdivi-1 for 24 h, then seeded into Matrigel to induce tube formation under normal or hypoxic conditions. Tube structure was observed at 18 h. (F) Total tube length was measured. All experiments were performed in triplicates. *p<0.05, NS, no significant.

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  Fig. 5 DRP1 silencing inhibits hypoxia-induced angiogenic function. (A) EPCs were transiently induced with DRP1 siRNA for 48 h. Cells were harvested and detected the expression of DRP1 to confirm silencing. DRP1 siRNA dose-dependent transduction showed decreased expression. (B) DRP1 silencing cells were seeded into the upper chamber with or without hypoxia for 6 h. DRP1 silencing cells showed decreased cell migration ability with or without hypoxic condition. (C) DRP1 silencing cells were seeded into Matrigel-coated transwell, and incubated under normoxic or hypoxic condition for 24 h. (D, E) Migrating (D) and Invading cells (E) were quantified using the Image J software. All experiment were conducted in at least triplicates. *p<0.05, **p<0.01. (F) DRP1 silencing attenuated the ability of tube formation in the hypoxic condition. Tube structure was observed at 18 h. (G) Total tube length was measured using the Image J software. All experiment were performed at least triplicates. *p<0.05.

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  Fig. 6 Mitochondrial dynamics does not influence the expression of EPC surface markers. The effect of Mdivi-1 on the expression of EPCs surface marker was observed using flow cytometry analysis. EPCs were cultured with or without 12.5 µM Mdivi-1 for 24 h under hypoxia or normoxia; expression of surface markers CD34 and C-kit/CD31/KDR/CXCR4 was analyzed.

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