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The modulating effects of the overexpression of uncoupling protein 2 on the formation of reactive oxygen species in vascular cells
Diabetes Research and Clinical Practice 77S (2007) S46–S48 www.elsevier.com/locate/diabres
The modulating effects of the overexpression of uncoupling protein 2 on the formation of reactive oxygen species in vascular cells§ Hye-Soon Kim a, Keun-Gyu Park a, Tae Bon Koo b, Seung Huh c, In-Kyu Lee d,* a Department of Internal Medicine, Keimyung University School of Medicine, Daegu, Korea Department of Obstetrics and Gynecology, Kyungpook National Unieversity School of Medicine, Korea c Department of Vascular Surgery, Kyungpook National Unieversity School of Medicine, Korea d Department of Internal Medicine, Kyungpook National University School of Medicine, #50 Samduk-2Ga, Jung-Gu, Daegu 700-721, Korea b
Accepted 29 January 2007 Available online 25 April 2007
Abstract Uncoupling protein 2 (UCP-2) is a newly identified member of the mitochondrial anion carrier family and shares 60% sequence identity with the well-characterized thermogenic UCP-1 from brown adipose tissue. Several lines of evidence suggest that UCP-2 is involved in the control of reactive oxygen species (ROS) production by mitochondria. More recently, a direct role for UCP-2 in the regulation of atherogenesis has been suggested by the observation that bone marrow transplantation from UCP-2-deficient mice to low-density lipoprotein receptor-deficient mice markedly increased atherosclerotic lesion size. This review introduces the possible role of UCP-2 in the regulation of atherogenesis in vascular cells. Although the relative contribution of the individual ROS generating systems in the vasculature is still ambiguous, both cell membrane NAD(P)H oxidase and the mitochondrial electrontransport chain have been proposed to play significant roles in the overproduction of ROS. UCP-2 can possibly modify atherosclerotic processes initiated in vascular cells and agents that increase UCP-2 expression in vascular cells may help prevent the development and progression of atherosclerosis in patients with diabetes or hypertension. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: UCP-2; ROS; Vascular cells; Atherosclerosis
1. Introduction Overproduction of superoxide by the mitochondrial electron-transport chain has been implicated in the pathogenesis of vascular complications in patients with diabetes mellitus [1]. Uncoupling protein 2 (UCP-2), a § Presented in 13th Korea–Japan Symposium on Diabetes Mellitus which was held on November 11–12, 2005 in Seoul, Korea. * Corresponding author. Tel.: +82 53 420 5564; fax: +82 53 250 8010. E-mail address: [email protected] (I.-k. Lee).
member of the mitochondrial anion carrier family, shares 60% sequence identity with the brown fatspecific mitochondrial inner membrane protein UCP-1, and is expressed in a number of tissues [2]. UCP-2 dissipates the proton electrochemical gradient across the inner mitochondrial membrane through an uncoupling process [3] and is involved in the control of reactive oxygen species (ROS) generation by mitochondria [4]. Transplantation of UCP-2-deficient (UCP2 / ) bone marrow cells to low-density lipoprotein receptor-deficient (LDLR / ) mice markedly accelerated the formation of atherosclerotic lesions and
0168-8227/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2007.01.032
H.-S. Kim et al. / Diabetes Research and Clinical Practice 77S (2007) S46–S48
arterial walls isolated from the mice had increased macrophage accumulation in the atherosclerotic plaques [5]. These findings suggest a direct role for UCP-2 in the regulation of atherogenesis. In this review, the role of UCP-2 in development of atherosclerosis was discussed in vascular smooth muscle cells (VSCMs), endothelial cells (ECs), and monocytes/macrophages. 2. The effects of UCP-2 on VSMCs UCP-2 is basally expressed in VSMCs and is upregulated by Ang II and/or high glucose, both of which are thought to play major roles in the development of atherosclerosis. Overexpression of the UCP-2 gene by adenoviral vector transfer to VSMCs profoundly reduced ROS generation in response to high glucose and Ang II. UCP-2 overexpression also reduced AP-1 activity and mRNA expression of PAI-1, and inhibited proliferation and migration of human VSMCs in response to high glucose and Ang II [6]. We previously demonstrated that inhibition of AP-1 suppressed high glucose- and Ang-II-induced cellular proliferation and PAI-1 gene expression in VSMCs [7,8]. These results suggest that high glucose and Ang II stimulate ROS generation and subsequent activation of AP-1, which is correlated with the increased PAI-1 expression and proliferation/migration of VSMCs. The human UCP-2 promoter region has AP-1 binding sites near the transcription initiation site [9], which suggests that intracellular ROS-induced activation of AP-1 can upregulate UCP-2 expression. Increases in UCP-2 may, in turn, decrease ROS generation and AP-1 activity. Thus, high glucose- or Ang II-induced UCP-2 overexpression may be a compensatory mechanism for reducing ROS generation and limiting detrimental cellular effects of AP-1 on PAI-1 expression and cellular proliferation/migration in VSMCs. 3. The effects of UCP-2 on ECs The endothelium is important in the regulation of VSMCs and modulates vascular tone by releasing factors which promote relaxation, such as nitric oxide (NO) and prostacyclin, as well as components which induce contraction, such as endothlin-1 (ET-1) [10,11]. UCP-2 is expressed in ECs, and is upregulated in response to lysophosphatidylcholine (LPC) and linoleic acid, which cause mitochondrial calcium accumulation, transient plasma membrane hyperpolarization, and apoptosis of ECs [12]. Overexpression of the UCP-2 gene by adenoviral vector transfection of ECs profoundly reduced ROS generation, NF-kB activation,
S47
and mRNA expression of ET-1 in response to LPC and linoleic acid. UCP-2 also enhanced eNOS transcription, reduced LPC-induced impairment of endotheliumdependent vascular relaxation, and inhibited apoptosis of ECs in response to LPC and linoleic acid. The induction of UCP-2 by ROS may be a compensatory mechanism to overcome increased oxidative stress, but LPL or linoliec acid induced increased expression of UCP-2 mRNA may not be sufficient to protect against this stress. However, high levels of UCP-2 overexpression, produced by high doses of adenoviral vector (6 106 pfu), almost completely suppressed LPL and linoleic acid induced ROS generation. In contrast, suppression of endogenous expression of UCP-2 using a UCP-2-specific siRNA exaggerated linoleic acid and LPC-induced apoptosis of ECs. These results collectively suggest that UCP-2 functions as a physiological regulator of ROS generation and that overexpression of UCP-2 in ECs may contribute to prevention of atherosclerosis. 4. The effects of UCP-2 on monocytes Macrophages, which are derived from monocytes that are recruited to and accumulate in the early atherosclerotic lesion, play a pivotal role in the development of atherosclerosis. Monocyte integrins recognize vascular cell adhesion molecules on endothelial cells and mediate monocyte adhesion to ECs. Endogenous UCP-2 expression in monocytes is very low indicating a relatively minor role of UCP-2 under basal conditions. In contrast, H2O2 rapidly induces UCP-2 expression in monocytes, suggesting a role of UCP-2 in response to oxidative insults [13]. Overexpression of UCP-2 in monocytes reduced both basal and H2O2-induced ROS generation. Cellular redox state is thought to play a major role in the expression of b2 integrins and adhesion molecules in inflammatory cells, and the endogenous oxidant H2O2 activates CD11b/ CD18-dependent cell adhesion [14]. UCP-2 overexpression reduces expression of basal and H2O2induced b2 integrin, CCR2, and adhesion molecules such as ICAM-1 and VCAM-1. UCP-2 overexpression also reduced H2O2-induced calcium influx as well as steady-state cytosolic calcium levels, but not ATPinduced calcium influx and b2 integrin expression, suggesting that decreased ROS production by UCP-2 is responsible for the reduction in calcium influx and consequent decline in b2 integrin expression. Thus, UCP-2 overexpression inhibits b2 integrin-mediated firm adhesion to endothelial layers as well as inhibiting cell spreading and actin polymerization in response to
S48
H.-S. Kim et al. / Diabetes Research and Clinical Practice 77S (2007) S46–S48
Program of the Ministry of Commerce, Industry and Energy.
References
Fig. 1. Antiatherogenic effects of UCP-2 overexpression in vascular cells.
TNF-a and MCP-1. These results suggest that intracellular ROS production determines the functional activity of monocytes, and that UCP-2 overexpression in monocytes was able to reduce mitochondrial ROS generation, inhibit b2 integrin-mediated tight adhesion, and may decrease transendothelial migration of circulating monocytes, ultimately providing an anti-atherogenic effect. 5. Conclusions Increased ROS production contributes to the pathogenesis of atherosclerosis. Whereas UCP-1 is expressed only in brown adipose tissue, UCP-2 is expressed in many tissues, including vascular cells. Endogenous UCP-2 expression in vascular cells is very low under resting conditions. However, oxidative stresses can rapidly induce UCP-2 expression. Induction of UCP-2 by ROS may be a compensatory mechanism to overcome increased oxidative stress, but ROS-induced increased expression of UCP-2 may not be sufficient to protect cells against oxidative stress. However, a high level of UCP-2 overexpression successfully protects vascular cells against formation of atherosclerotic lesions (Fig. 1). These results present the possibility that agents which may increase UCP-2 expression in the vasculature may prevent the development and progression of atherosclerosis in patients with increased ROS, such as in diabetes and hypertension. Acknowledgements This study was supported by the Korea Science and Engineering Foundation through the NRL program (grant M106 00000271-06J000-27110) and by grant no. RTI04-01-01 from Regional Technology Innovation
[1] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813–820. [2] O. Boss, T. Hagen, B.B. Lowell, Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism, Diabetes 49 (2000) 143–156. [3] Y. Teshima, M. Akao, S.P. Jones, E. Marban, Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes, Circ. Res. 93 (2003) 192–200. [4] D. Arsenijevic, H. Onuma, C. Pecqueur, S. Raimbault, B.S. Manning, B. Miroux, et al., Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production, Nat. Genet. 26 (2000) 435–439. [5] J. Blanc, M.C. Alves-Guerra, B. Esposito, S. Rousset, P. Gourdy, D. Ricquier, et al., Protective role of uncoupling protein 2 in atherosclerosis, Circulation 107 (2003) 388–390. [6] J.Y. Park, K.G. Park, H.J. Kim, H.G. Kang, J.D. Ahn, H.S. Kim, et al., The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells, Diabetologia 48 (2005) 1022–1028. [7] J.D. Ahn, R. Morishita, Y. Kaneda, K.U. Lee, J.Y. Park, Y.J. Jeon, et al., Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells, Diabetologia 44 (2001) 713–720. [8] J.D. Ahn, R. Morishita, Y. Kaneda, S.J. Lee, K.Y. Kwon, S.Y. Choi, et al., Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo, Circ. Res. 90 (2002) 1325–1332. [9] N. Tu, H. Chen, U. Winnikes, I. Reinert, G. Marmann, K.M. Pirke, et al., Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene, Biochem. Biophys. Res. Commun. 265 (1999) 326–334. [10] J.C. Choy, D.J. Granville, D.W. Hunt, B.M. McManus, Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis, J. Mol. Cell. Cardiol. 33 (2001) 1673–1690. [11] J.V. Mombouli, P.M. Vanhoutte, Endothelial dysfunction: from physiology to therapy, J. Mol. Cell. Cardiol. 31 (1999) 61–74. [12] K.U. Lee, I.K. Lee, J. Han, D.K. Song, Y.M. Kim, H.S. Song, et al., Effects of recombinant adenovirus-mediated uncoupling protein 2 overexpression on endothelial function and apoptosis, Circ. Res. 96 (2005) 1200–1207. [13] J.W. Ryu, K.H. Hong, J.H. Maeng, J.B. Kim, J. Ko, Y.Y. Park, et al., Overexpression of uncoupling protein 2 in THP1 monocytes inhibits beta2 integrin-mediated firm adhesion and transendothelial migration, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 864–870. [14] G. Skoglund, I. Cotgreave, J. Rincon, M. Patarroyo, M. Ingelman-Sundberg, H2O2 activates CD11b/CD18-dependent cell adhesion, Biochem. Biophys. Res. Commun. 157 (1988) 443– 449.
The modulating effects of the overexpression of uncoupling protein 2 on the formation of reactive oxygen species in vascular cells§ Hye-Soon Kim a, Keun-Gyu Park a, Tae Bon Koo b, Seung Huh c, In-Kyu Lee d,* a Department of Internal Medicine, Keimyung University School of Medicine, Daegu, Korea Department of Obstetrics and Gynecology, Kyungpook National Unieversity School of Medicine, Korea c Department of Vascular Surgery, Kyungpook National Unieversity School of Medicine, Korea d Department of Internal Medicine, Kyungpook National University School of Medicine, #50 Samduk-2Ga, Jung-Gu, Daegu 700-721, Korea b
Accepted 29 January 2007 Available online 25 April 2007
Abstract Uncoupling protein 2 (UCP-2) is a newly identified member of the mitochondrial anion carrier family and shares 60% sequence identity with the well-characterized thermogenic UCP-1 from brown adipose tissue. Several lines of evidence suggest that UCP-2 is involved in the control of reactive oxygen species (ROS) production by mitochondria. More recently, a direct role for UCP-2 in the regulation of atherogenesis has been suggested by the observation that bone marrow transplantation from UCP-2-deficient mice to low-density lipoprotein receptor-deficient mice markedly increased atherosclerotic lesion size. This review introduces the possible role of UCP-2 in the regulation of atherogenesis in vascular cells. Although the relative contribution of the individual ROS generating systems in the vasculature is still ambiguous, both cell membrane NAD(P)H oxidase and the mitochondrial electrontransport chain have been proposed to play significant roles in the overproduction of ROS. UCP-2 can possibly modify atherosclerotic processes initiated in vascular cells and agents that increase UCP-2 expression in vascular cells may help prevent the development and progression of atherosclerosis in patients with diabetes or hypertension. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: UCP-2; ROS; Vascular cells; Atherosclerosis
1. Introduction Overproduction of superoxide by the mitochondrial electron-transport chain has been implicated in the pathogenesis of vascular complications in patients with diabetes mellitus [1]. Uncoupling protein 2 (UCP-2), a § Presented in 13th Korea–Japan Symposium on Diabetes Mellitus which was held on November 11–12, 2005 in Seoul, Korea. * Corresponding author. Tel.: +82 53 420 5564; fax: +82 53 250 8010. E-mail address: [email protected] (I.-k. Lee).
member of the mitochondrial anion carrier family, shares 60% sequence identity with the brown fatspecific mitochondrial inner membrane protein UCP-1, and is expressed in a number of tissues [2]. UCP-2 dissipates the proton electrochemical gradient across the inner mitochondrial membrane through an uncoupling process [3] and is involved in the control of reactive oxygen species (ROS) generation by mitochondria [4]. Transplantation of UCP-2-deficient (UCP2 / ) bone marrow cells to low-density lipoprotein receptor-deficient (LDLR / ) mice markedly accelerated the formation of atherosclerotic lesions and
0168-8227/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2007.01.032
H.-S. Kim et al. / Diabetes Research and Clinical Practice 77S (2007) S46–S48
arterial walls isolated from the mice had increased macrophage accumulation in the atherosclerotic plaques [5]. These findings suggest a direct role for UCP-2 in the regulation of atherogenesis. In this review, the role of UCP-2 in development of atherosclerosis was discussed in vascular smooth muscle cells (VSCMs), endothelial cells (ECs), and monocytes/macrophages. 2. The effects of UCP-2 on VSMCs UCP-2 is basally expressed in VSMCs and is upregulated by Ang II and/or high glucose, both of which are thought to play major roles in the development of atherosclerosis. Overexpression of the UCP-2 gene by adenoviral vector transfer to VSMCs profoundly reduced ROS generation in response to high glucose and Ang II. UCP-2 overexpression also reduced AP-1 activity and mRNA expression of PAI-1, and inhibited proliferation and migration of human VSMCs in response to high glucose and Ang II [6]. We previously demonstrated that inhibition of AP-1 suppressed high glucose- and Ang-II-induced cellular proliferation and PAI-1 gene expression in VSMCs [7,8]. These results suggest that high glucose and Ang II stimulate ROS generation and subsequent activation of AP-1, which is correlated with the increased PAI-1 expression and proliferation/migration of VSMCs. The human UCP-2 promoter region has AP-1 binding sites near the transcription initiation site [9], which suggests that intracellular ROS-induced activation of AP-1 can upregulate UCP-2 expression. Increases in UCP-2 may, in turn, decrease ROS generation and AP-1 activity. Thus, high glucose- or Ang II-induced UCP-2 overexpression may be a compensatory mechanism for reducing ROS generation and limiting detrimental cellular effects of AP-1 on PAI-1 expression and cellular proliferation/migration in VSMCs. 3. The effects of UCP-2 on ECs The endothelium is important in the regulation of VSMCs and modulates vascular tone by releasing factors which promote relaxation, such as nitric oxide (NO) and prostacyclin, as well as components which induce contraction, such as endothlin-1 (ET-1) [10,11]. UCP-2 is expressed in ECs, and is upregulated in response to lysophosphatidylcholine (LPC) and linoleic acid, which cause mitochondrial calcium accumulation, transient plasma membrane hyperpolarization, and apoptosis of ECs [12]. Overexpression of the UCP-2 gene by adenoviral vector transfection of ECs profoundly reduced ROS generation, NF-kB activation,
S47
and mRNA expression of ET-1 in response to LPC and linoleic acid. UCP-2 also enhanced eNOS transcription, reduced LPC-induced impairment of endotheliumdependent vascular relaxation, and inhibited apoptosis of ECs in response to LPC and linoleic acid. The induction of UCP-2 by ROS may be a compensatory mechanism to overcome increased oxidative stress, but LPL or linoliec acid induced increased expression of UCP-2 mRNA may not be sufficient to protect against this stress. However, high levels of UCP-2 overexpression, produced by high doses of adenoviral vector (6 106 pfu), almost completely suppressed LPL and linoleic acid induced ROS generation. In contrast, suppression of endogenous expression of UCP-2 using a UCP-2-specific siRNA exaggerated linoleic acid and LPC-induced apoptosis of ECs. These results collectively suggest that UCP-2 functions as a physiological regulator of ROS generation and that overexpression of UCP-2 in ECs may contribute to prevention of atherosclerosis. 4. The effects of UCP-2 on monocytes Macrophages, which are derived from monocytes that are recruited to and accumulate in the early atherosclerotic lesion, play a pivotal role in the development of atherosclerosis. Monocyte integrins recognize vascular cell adhesion molecules on endothelial cells and mediate monocyte adhesion to ECs. Endogenous UCP-2 expression in monocytes is very low indicating a relatively minor role of UCP-2 under basal conditions. In contrast, H2O2 rapidly induces UCP-2 expression in monocytes, suggesting a role of UCP-2 in response to oxidative insults [13]. Overexpression of UCP-2 in monocytes reduced both basal and H2O2-induced ROS generation. Cellular redox state is thought to play a major role in the expression of b2 integrins and adhesion molecules in inflammatory cells, and the endogenous oxidant H2O2 activates CD11b/ CD18-dependent cell adhesion [14]. UCP-2 overexpression reduces expression of basal and H2O2induced b2 integrin, CCR2, and adhesion molecules such as ICAM-1 and VCAM-1. UCP-2 overexpression also reduced H2O2-induced calcium influx as well as steady-state cytosolic calcium levels, but not ATPinduced calcium influx and b2 integrin expression, suggesting that decreased ROS production by UCP-2 is responsible for the reduction in calcium influx and consequent decline in b2 integrin expression. Thus, UCP-2 overexpression inhibits b2 integrin-mediated firm adhesion to endothelial layers as well as inhibiting cell spreading and actin polymerization in response to
S48
H.-S. Kim et al. / Diabetes Research and Clinical Practice 77S (2007) S46–S48
Program of the Ministry of Commerce, Industry and Energy.
References
Fig. 1. Antiatherogenic effects of UCP-2 overexpression in vascular cells.
TNF-a and MCP-1. These results suggest that intracellular ROS production determines the functional activity of monocytes, and that UCP-2 overexpression in monocytes was able to reduce mitochondrial ROS generation, inhibit b2 integrin-mediated tight adhesion, and may decrease transendothelial migration of circulating monocytes, ultimately providing an anti-atherogenic effect. 5. Conclusions Increased ROS production contributes to the pathogenesis of atherosclerosis. Whereas UCP-1 is expressed only in brown adipose tissue, UCP-2 is expressed in many tissues, including vascular cells. Endogenous UCP-2 expression in vascular cells is very low under resting conditions. However, oxidative stresses can rapidly induce UCP-2 expression. Induction of UCP-2 by ROS may be a compensatory mechanism to overcome increased oxidative stress, but ROS-induced increased expression of UCP-2 may not be sufficient to protect cells against oxidative stress. However, a high level of UCP-2 overexpression successfully protects vascular cells against formation of atherosclerotic lesions (Fig. 1). These results present the possibility that agents which may increase UCP-2 expression in the vasculature may prevent the development and progression of atherosclerosis in patients with increased ROS, such as in diabetes and hypertension. Acknowledgements This study was supported by the Korea Science and Engineering Foundation through the NRL program (grant M106 00000271-06J000-27110) and by grant no. RTI04-01-01 from Regional Technology Innovation
[1] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 414 (2001) 813–820. [2] O. Boss, T. Hagen, B.B. Lowell, Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism, Diabetes 49 (2000) 143–156. [3] Y. Teshima, M. Akao, S.P. Jones, E. Marban, Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes, Circ. Res. 93 (2003) 192–200. [4] D. Arsenijevic, H. Onuma, C. Pecqueur, S. Raimbault, B.S. Manning, B. Miroux, et al., Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production, Nat. Genet. 26 (2000) 435–439. [5] J. Blanc, M.C. Alves-Guerra, B. Esposito, S. Rousset, P. Gourdy, D. Ricquier, et al., Protective role of uncoupling protein 2 in atherosclerosis, Circulation 107 (2003) 388–390. [6] J.Y. Park, K.G. Park, H.J. Kim, H.G. Kang, J.D. Ahn, H.S. Kim, et al., The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells, Diabetologia 48 (2005) 1022–1028. [7] J.D. Ahn, R. Morishita, Y. Kaneda, K.U. Lee, J.Y. Park, Y.J. Jeon, et al., Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose- and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells, Diabetologia 44 (2001) 713–720. [8] J.D. Ahn, R. Morishita, Y. Kaneda, S.J. Lee, K.Y. Kwon, S.Y. Choi, et al., Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo, Circ. Res. 90 (2002) 1325–1332. [9] N. Tu, H. Chen, U. Winnikes, I. Reinert, G. Marmann, K.M. Pirke, et al., Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene, Biochem. Biophys. Res. Commun. 265 (1999) 326–334. [10] J.C. Choy, D.J. Granville, D.W. Hunt, B.M. McManus, Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis, J. Mol. Cell. Cardiol. 33 (2001) 1673–1690. [11] J.V. Mombouli, P.M. Vanhoutte, Endothelial dysfunction: from physiology to therapy, J. Mol. Cell. Cardiol. 31 (1999) 61–74. [12] K.U. Lee, I.K. Lee, J. Han, D.K. Song, Y.M. Kim, H.S. Song, et al., Effects of recombinant adenovirus-mediated uncoupling protein 2 overexpression on endothelial function and apoptosis, Circ. Res. 96 (2005) 1200–1207. [13] J.W. Ryu, K.H. Hong, J.H. Maeng, J.B. Kim, J. Ko, Y.Y. Park, et al., Overexpression of uncoupling protein 2 in THP1 monocytes inhibits beta2 integrin-mediated firm adhesion and transendothelial migration, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 864–870. [14] G. Skoglund, I. Cotgreave, J. Rincon, M. Patarroyo, M. Ingelman-Sundberg, H2O2 activates CD11b/CD18-dependent cell adhesion, Biochem. Biophys. Res. Commun. 157 (1988) 443– 449.