Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

Kwon Ho Lee; Sang Ki Lee; Hyo Shin Kim; Eun Jung Cho; Hee Kyoung Joo; Eun Ji Lee; Ji Young Lee; Myoung Soo Park; Seok Jong Chang; Chung-Hyun Cho; Jin Bong Park; Byeong Hwa Jeon

doi:10.4196/kjpp.2009.13.6.431

Journal List > Korean J Physiol Pharmacol > v.13(6) > 1025638

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Lee, Lee, Kim, Cho, Joo, Lee, Lee, Park, Chang, Cho, Park, and Jeon: Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

Original Article

Korean J Physiol Pharmacol 2009;13(6):431-436.

Published online: 18 February 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.6.431

Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

Kwon Ho Lee^1,^∗, Sang Ki Lee^1,^∗, Hyo Shin Kim^1,², Eun Jung Cho^1,², Hee Kyoung Joo^1,², Eun Ji Lee^1,², Ji Young Lee^1,², Myoung Soo Park^1,², Seok Jong Chang⁴, Chung-Hyun Cho^1,^2,³, Jin Bong Park^1,², Byeong Hwa Jeon^1,^2,^3,

¹Department of Physiology, Chungnam National University, Daejeon 301-131, Korea

²Department of Infection Signaling Network Research Center, Chungnam National University, Daejeon 301-131, Korea

³Department of Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon 301-131, Korea

⁴Department of Physiology, College of Medicine, Seonam University, Namwon 590-711, Korea

Corresponding to: Byeong Hwa Jeon, Infection Signaling Network Research Center, Department of Physiology, School of Medicine, Chungnam National University, 6, Munhwa-dong, Jung-gu, Daejeon 301-131, Korea. (Tel) 82-42-580-8214, (Fax) 82-42-585-8440, (E-mail) bhjeon@cnu.ac.kr

^∗ First two authors contributed equally to this article.

Received 13 October 2009 Revised 9 November 2009 Accepted 10 November 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

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Keywords: Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1), Lead, Cell death, Endothelial cells, Catalase

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Fig. 1.

Effect of lead (Pb) on endothelial cell viability. (A) Typical endothelial cell morphology after treatment with Pb acetate (30 μM) for 12 ∼48 hr (a, control; b, 12 hr; c, 24 hr; d, 48 hr). (B) Summarized data are plotted in Fig. 1B. (C) Typical endothelial cell morphology after treatment with Pb acetate for 24 hr. (a, control; b, 3 μM of Pb; c, 10 μM of Pb; d, 30 μM of Pb; e, 100 μM of Pb; f, 300 μM of Pb). (D) Summarized data was plotted in Fig. 1D. Total acetate concentration (300 μM) was balanced with Na acetate in Fig. 1C. Endothelial cell viability was measured via a MTT assay as described in the Materials and Methods section. The data are expressed as the means± SEM for 3 separate experiments. ^∗p <0.05 compared with control.

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Fig. 2.

Ref-1 overexpression inhibited lead (Pb) induced endothelial cell death. (A) Endothelial cell morphology in the Pb (100 μM)-treated endothelial cells which were transfected with Adβgal or AdRef-1. Adenoviral transfection of AdRef-1 successfully overexpressed Ref-1 in the endothelial cells as compared with Adβgal. (B) Ref-1 overexpression induced by AdRef-1 inhibited Pb (100 μM)-induced endothelial cell death (in the bottom of Fig. 2A). (B) Summarized data was plotted in Fig. 2B. Endothelial cell viability was measured with an MTT assay as described in the Materials and Methods section. The data are expressed as the means± SEM for 4 separate experiments. ^∗p < 0.05 compared with control cells. ^#p<0.05 compared with Adβgal+Pb.

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Fig. 3.

Ref-1 overexpression inhibited lead (Pb)-induced superoxide and hydrogen peroxide production. Pb (100 μM) was administered for 24 hr in the endothelial cells transfected with Adβgal or AdRef-1. Intracellular superoxide (A) and hydrogen peroxide production (C) were evaluated using the superoxide-sensitive fluorophore dihydroethidine (DHE) and the peroxide-sensitive fluorophore 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). (B, D) The summarized data was plotted in Figs. 3B and 3D. The data are expressed as the means± SEM for 3 separate experiments. ^∗p <0.05 compared with Adβgal + Pb.

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Fig. 4.

Ref-1 inhibited lead-induced catalase suppression in the endothelial cells. (A) Lead (Pb) treatment resulted in the downregulation of catalase expression. Endothelial cells were exposed to lead (3∼300 μM) for 24 hr. Western blot analysis for catalase expression was conducted. β-actin was used as a loading control. (B) Summarized data was plotted in Fig. 4B. ^ap<0.05 compared with control. The data were expressed as the means±SEM for 3 separate experiments. (C) Effect of Ref-1 on the Pb-induced suppression of catalase expression in the endothelial cells. Endothelial cells were exposed by lead (30 μM) for 24 hr in the endothelial cells transfected with 20 or 200 MOI of AdRef-1. A total of 200 MOI of adenoviral transfection was balanced with Adβgal. (D) The summarized data was plotted in Fig. 4D. The data were expressed as the means± SEM for 3 separate experiments. ^ap <0.05 compared with Adβgal 200 MOI, ^bp<0.05 compared with AdβGal + Pb, and ^cp<0.05 compared with AdRef-1 20 MOI + Pb.

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