Korean Circ J.  2010 Dec;40(12):651-658. 10.4070/kcj.2010.40.12.651.

Expression Pattern of the Thioredoxin System in Human Endothelial Progenitor Cells and Endothelial Cells Under Hypoxic Injury

• Park KJ
• Kim YJ
• Choi EJ
• Park NK
• Kim GH
• Kim SM
• Lee SY
• Bae JW
• Hwang KK
• Kim DW
• Cho MC

• KMID: 2225164
• DOI: http://doi.org/10.4070/kcj.2010.40.12.651

Abstract

BACKGROUND AND OBJECTIVES
The thioredoxin (TRx) system is a ubiquitous thiol oxidoreductase pathway that regulates cellular reduction/oxidation status. Although endothelial cell (EC) hypoxic damage is one of the important pathophysiologic mechanisms of ischemic heart disease, its relationship to the temporal expression pattern of the TRx system has not yet been elucidated well. The work presented here was performed to define the expression pattern of the TRx system and its correlation with cellular apoptosis in EC lines in hypoxic stress. These results should provide basic clues for applying aspects of the TRx system as a therapeutic molecule in cardiovascular diseases.
SUBJECTS AND METHODS
Hypoxia was induced with 1% O2, generated in a BBL GasPak Pouch (Becton Dickinson, Franklin Lakes, NJ, USA) in human endothelial progenitor cells (hEPC) and human umbilical vein endothelial cells (HUVEC). Apoptosis of these cells was confirmed by Annexin-V: Phycoerythrin flow cytometry. Expression patterns of TRx; TRx reductase; TRx interacting protein; and survival signals, such as Bcl-2 and Bax, in ECs under hypoxia were checked.
RESULTS
Apoptosis was evident after hypoxia in the two cell types. Higher TRx expression was observed at 12 hours after hypoxia in hEPCs and 12, 36, 72 hours of hypoxia in HUVECs. The expression patterns of the TRx system components showed correlation with EC apoptosis and cell survival markers.
CONCLUSION
Hypoxia induced significant apoptosis and its related active changes of the TRx system were evident in human EC lines. If the cellular impact of TRx expression pattern in various cardiovascular tissues under hypoxia or oxidative stress was studied meticulously, the TRx system could be applied as a new therapeutic target in cardiovascular diseases, such as ischemic heart disease or atherosclerosis.

Keyword

Thioredoxins; Apoptosis; Endothelial cells; Cell hypoxia

MeSH Terms

Anoxia
Apoptosis
Atherosclerosis
Cardiovascular Diseases
Cell Hypoxia
Cell Survival
Endothelial Cells
Flow Cytometry
Human Umbilical Vein Endothelial Cells
Humans
Lakes
Myocardial Ischemia
Oxidative Stress
Phycoerythrin
Stem Cells
Thioredoxins
Phycoerythrin
Thioredoxins

Figure

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  Fig. 1 Apoptosis in hEPC and HUVEC during hypoxia via FACS analysis. Hypoxia generated by the GasPak system, hEPC showed bimodal peaks of apoptosis at 12 and 24 hours (175.9±35.8%, 398.3±75.8%), 60 and 72 hours (220.5±68.1%, 368.8±20.3%) of hypoxia. HUVECs showed increased apoptosis at 12 hours (256.3±42.2%) of hypoxia initially and the highest degree of apoptosis was observed at 72 hours (500.6±45.6%) of hypoxia. *Significantly different from the control time (p<0.05). hEPC: human endothelial progenitor cells, HUVEC: human umbilical vein endothelial cells, FACS: fluorescence-activated cell sorting.

• 

  Fig. 2 The expression pattern of the TRx system in hEPCs and HUVECs during hypoxia. In hEPCs during hypoxia, TRx expression was elevated at 12 and 24 hours (651.3±78.5%, 593.5±64.3%) and thereafter. In HUVECs, TRx expression was increased at 12, 36, and 72 hours of hypoxia (640.1±89.4%, 620.2±98.1%, 595.9±128.4% on each time point), but the over-expression of TRxR and TxNip was not observed during whole hypoxic period. The TRx/TxNip ratio was increased at 36 hours (250.8±77.8%) of hypoxia in hEPCs and at 48 and 72 hours (301.7±49.2%, 550.4±78.4%) of hypoxia in HUVECs. *Significantly different from the control time (p<0.05). TRx: thioredoxin, hEPC: human endothelial progenitor cells, HUVEC: human umbilical vein endothelial cells, TRxR: TRx reductase, TxNip: TRx interacting protein.

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  Fig. 3 TRx mRNA expression in hEPCs and HUVECs during hypoxic stress. TRx mRNA expression was increased significantly from 24 hours and thereafter during hypoxia in hEPCs (193.5±56.3%, 188.7±65.9%, 205.9±61.2% on 24, 36, 47 hours), but no increase in TRx mRNA expression in HUVECs was evident during the whole hypoxic period. *Significantly different from the control time (p<0.05). TRx: thioredoxin, mRNA: messenger ribonucleic acid, hEPC: human endothelial progenitor cells, HUVEC: human umbilical vein endothelial cells.

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  Fig. 4 The expression pattern of Bax and Bcl-2 in hEPCs and HUVECs during hypoxia. In hEPC, Bcl-2 expression showed 2 peaks at 12 and 60 hours (182.7±48.3%, 189.5±52.9%) of hypoxia, Bax expression was not increased significantly. In HUVECs, there were 3 peaks of Bcl-2 expression at 6 to 12, 36, and 60 to 72 hours (259.4±44.9%, 243.4±38.7%, 274.8±46.8%, 220.9±49.7%, 279.3±51.1% on each time point) of hypoxia and the Bax expression pattern was similar. Higher values of the Bax/Bcl-2 ratio were observed at 24 hours (212.4±67.0% vs. 110.8±48.3%) and 60 hours (275.9±86.3% vs. 89.5±59.1%) of hypoxia in hEPCs compared with HUVECs. *Significantly different from the control time (p<0.05), †Significantly different between hEPC and HUVEC (p<0.05). hEPC: human endothelial progenitor cells, HUVEC: human umbilical vein endothelial cells.

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  Fig. 5 The expression pattern of Caspase 3, Akt, and Survivin in hEPCs and HUVECs during hypoxia. A steep increase in the expression of Akt was observed in hEPC and HUVECs. Akt overexpression was significantly increased compared with control starting at 12 hours of hypoxia in hEPCs and starting at 24 hours of hypoxia in HUVECs. Caspase 3 and survivin activation was not evident in hEPCs during whole hypoxic period. Caspase 3 overexpression in HUVECs was observed starting at 48 hours (196.8±45.9%) of hypoxia. Higher survivin expression was observed at 12, 60, and 72 hours (385.7±75.2%, 345.6±89.1%, 678.7±95.7% on each time point) of hypoxia in hEPCs. *Significantly different from the control time (p<0.05). hEPC: human endothelial progenitor cells, HUVEC: human umbilical vein endothelial cells, mRNA: messenger ribonucleic acid.

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