This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 23/12/2178    most recent 01.ATV.0000099788.31333.DBv1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, Y. Articles by Delafontaine, P. Search for Related Content PubMed PubMed Citation Articles by Li, Y. Articles by Delafontaine, P. Related Collections Apoptosis Pathophysiology Growth factors/cytokines Mechanism of atherosclerosis/growth factors

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:2178.)
© 2003 American Heart Association, Inc.

Vascular Biology

Insulin-Like Growth Factor-1 Receptor Activation Inhibits Oxidized LDL-Induced Cytochrome C Release and Apoptosis via the Phosphatidylinositol 3 Kinase/Akt Signaling Pathway

Yangxin Li; Yusuke Higashi; Hiroyuki Itabe; Yao-Hua Song; Jie Du; Patrice Delafontaine

From the Tulane University Medical Center, New Orleans, LA; Teikyo University, Kanagawa, Japan; and The University of Texas Medical Branch, Galveston, TX.

Correspondence to Patrice Delafontaine, MD, FACC, FAHA, FACP, FESC, Tulane University Medical Center, School of Medicine, Section of Cardiology, 1430 Tulane Avenue, New Orleans, LA 70112–2699. E-mail pdelafon{at}tulane.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— We have shown previously that oxidized LDL decreases insulin-like growth factor-1 (IGF-1) and IGF-1 receptor expression in vascular smooth muscle cells and that IGF-1 and IGF-1 receptor expression are reduced in the deep intima of early atherosclerotic lesions. Because oxidized LDL is potentially important for the depletion of vascular smooth muscle cells contributing to plaque destabilization, we studied the role of IGF-1 in oxidized LDL-induced apoptosis.

Methods and Results— We provide evidence that oxidized LDL-induced apoptosis is caused by decreased mitochondrial membrane potential and increased cytochrome C release in human aortic vascular smooth muscle cells. Overexpression of the IGF-1 receptor by using an adenovirus completely abrogated these effects. The antiapoptotic function of the IGF-1 receptor was associated with increased Akt kinase activity and increased expression of phosphorylated Bad. Moreover, a dominant-negative p85 phosphatidylinositol 3-kinase adenovirus blocked the capacity of the IGF-1 receptor to prevent oxidized LDL-induced apoptosis.

Conclusions— Our data demonstrate that IGF-1 receptor activation inhibits oxidized LDL-induced cytochrome C release and apoptosis through the phosphatidylinositol 3-kinase/Akt signaling pathway and suggest that genetic or pharmacological activation of the IGF-1 receptor may be a useful strategy to stabilize atherosclerotic plaques.

Key Words: vascular smooth muscle cells • atherosclerosis • growth factors • signal transduction • receptors

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atherosclerosis is the leading cause of mortality in the Western world. Oxidative modification of LDL is widely believed to be involved in the pathogenesis of atherosclerosis and in destabilization of the atherosclerotic plaque.^1–3 Foam cells, one of the hallmarks of atherosclerotic plaque, develop when monocyte-derived macrophages or vascular smooth muscle cells (VSMCs) within the arterial wall take up oxidized LDLs (oxLDLs) via scavenger receptors. OxLDLs can induce apoptosis of endothelial cells^4–6 and VSMCs⁷ in vitro, and oxLDLs are associated with the apoptosis of VSMCs in human atherosclerotic plaques.⁸ We have recently shown that oxLDLs reduce insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) expression in VSMCs⁹ and that IGF-1/IGF-1R expression is reduced in areas of atherosclerotic plaque.^8,10 Because of the potent survival effects of IGF-1,^11,12 these findings provide a putative mechanism whereby oxLDLs could contribute to plaque apoptosis.

Although the precise pathways mediating the survival action of IGF-1 are unclear, these effects are mediated through the type 1 IGF-1R, which possesses intrinsic tyrosine kinase activity and activates a number of downstream mediators, including phosphatidylinositol 3-kinase (PI3-kinase)/Akt and mitogen-activated protein kinase (MAPK).^13–16 Both MAPK and particularly PI -kinase/Akt have been implicated in IGF-1 and growth factor-induced survival effects.^16–18 The survival effects of Akt are mediated by phosphorylation and inhibition of several proapoptotic proteins, such as Bad,¹⁹ caspase 9,²⁰ and the Forkhead transcription factors.^11,21 Activation of Akt has been shown to prevent the release of cytochrome C from mitochondria,²² which occurs as a result of mitochondrial membrane permeabilization^23,24 and which is a critical event in both stress and death receptor–induced apoptosis.^25,26 On entry into the cytosol, cytochrome C binds the caspase-activating protein Apaf-1, stimulating its binding to procaspase 9 and activating caspase networks that trigger apoptosis.²⁷ There is limited information on the effect of oxLDL on mitochondrial function,^28,29 and the potential roles of oxLDLs mediated IGF-1 and IGF-1R downregulation in mitochondrial dysfunction and apoptosis are unknown.

In this study, we investigated the effects of oxLDLs on mitochondrial function, the release of cytochrome C and apoptosis in VSMCs, and the ability of IGF-1R activation to rescue cells from oxLDL-induced apoptosis. Our findings demonstrate that oxLDLs trigger loss of mitochondrial membrane potential and cytochrome C release, leading to apoptosis in VSMCs. Furthermore, overexpression of the IGF-1R completely rescues VSMCs from these effects through a PI3-kinase/Akt–dependent pathway. These findings have major implications for devising a strategy to limit the loss of VSMCs that contribute to the destabilization of advanced atherosclerotic plaque and could contribute to aneurysm rupture.^30,31

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
See online Materials and Methods, which can be accessed at http://atvb.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of oxLDL, IGF-1, and Overexpression of IGF-1R on Cell Viability
To determine the effect of IGF-1R overexpression on oxLDL-induced cytotoxicity, we incubated human airway smooth muscle cells (HASMCs) with native LDL (nLDL) or oxLDL (0 to 60 µg/mL) for 7, 16, or 24 hours in the presence or absence of IGF-1R adenovirus (AdIGF-1R). We found that oxLDL caused a reduction in HASMC viability at 24 hours but not at 7 and 16 hours, whereas nLDL did not affect cell viability (see online Figure IA, which can be accessed at http://atvb.ahajournals.org). In addition, the cells were transiently exposed to oxLDLs for 7 or 16 hours, the oxLDLs were then removed, and new culture media were added and toxicity determined at 24 hours after the initial exposure to oxLDLs. Cytotoxicity was induced after 16 but not 7 hours of exposure to oxLDLs (Figure IB). Both IGF-1 and des-(1,3)-IGF-1 (des-IGF-1) partially protected against oxLDL-induced cytotoxicity when added with oxLDL for 24 hours (Figure IC). The rationale for using des-IGF-1 is that it has markedly lower affinity for IGF binding proteins and that the effect of IGF-1 independent of IGF binding proteins can be assessed. However, overexpression of IGF-1R using AdIGF-1R totally protected HASMCs against oxLDL-induced loss of cell viability, whereas overexpression of the control adenovirus, AdGFP, had no protective effect (Figure ID). To determine the specificity of the effect of overexpressing the IGF-1R to prevent oxLDL-induced apoptosis, we performed additional experiments to determine whether other growth factors, namely basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF)-BB, can partially rescue VSMCs from the proapoptotic effect of oxLDLs. Because adenoviral constructs for these growth factor receptors are not available to us, we looked at the effect of adding exogenous growth factor to VSMCs in the presence of oxLDLs. As shown in the Figure 1, neither PDGF-BB nor bFGF rescued VSMCs from the effects of oxLDLs, whereas des-IGF-1 significantly blunted the effect of oxLDLs.

View larger version (24K): [in this window] [in a new window]   Figure 1. Effects of PDGF, bFGF, and des-IGF-1 on oxLDL-induced apoptosis. HASMCs were exposed to 60 µg/mL oxLDL alone (oxLDL) or with 50 ng/mL PDGF, bFGF, or des-IGF-1 for 24 hours and analyzed by death ELISA as described in the Materials and Methods. *P<0.05 compared with oxLDL alone.

To determine the principal components of oxLDL-induced cell death, we measured apoptosis by flow cytometry (Figure 2A) and by death ELISA (Figure 2B). OxLDLs caused a marked increase in Annexin-V staining (Figure 2A) and in antihistone and anti-DNA antibody binding (Figure 2B). Overexpression of the IGF-IR caused marked inhibition of oxLDL-induced apoptosis (Figure 2B).

View larger version (36K): [in this window] [in a new window]   Figure 2. A, Flow cytometry. HASMCs were incubated for 24 hours in serum-free medium alone or with 60 µg/mL nLDL or oxLDL and then stained for annexin V and propidium iodide, after which fluorescence-activated cell sorting analysis was performed immediately. Cells staining positive for Annexin V-FITC and negative for propidium iodide were considered as undergoing apoptosis. B, Death ELISA. HASMCs were infected with AdGFP or AdIGF-1R overnight and then exposed to serum-free medium alone (SFM) or with 60 µg/mL nLDL or oxLDL for 24 hours and analyzed for histone-associated DNA fragments as described in the Materials and Methods. **P<0.001 compared with AdGFP/nLDL; *P<0.005 compared with AdIGF-1R/nLDL; ¶P<0.01 compared with AdGFP/oxLDL.

Effects of oxLDLs and Overexpression of IGF-1R on
To determine the mechanisms by which oxLDLs induce apoptosis, we incubated HASMCs with various concentrations of oxLDLs. We found that oxLDLs caused a concentration and time-dependent decrease in and that a dose of 60 µg/mL oxLDL decreased at 7 hours but not at 2 hours (see online Figure II, which can be accessed at http://atvb.ahajournals.org). In contrast, nLDLs did not affect . Overexpression of IGF-1R using AdIGF-1R completely inhibited the oxLDL-induced loss of mitochondrial membrane potential. (Figure 3).

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of oxLDL and overexpression of IGF-1R on mitochondrial membrane potential. HASMCs were incubated alone (control) or with 60 µg/mL oxLDL for 7 hours or were infected with AdGFP or AdIGF-1R for 24 hours and then incubated with or without oxLDL (60 µg/mL) for 7 hours, washed with PBS 3 times, and mitochondrial membrane potential was measured using RH123 as described in the Materials and Methods. The results were expressed as a percentage of the control ± SEM. *P<0.01 compared with control, **P<0.01 compared with AdGFP.

Effects of oxLDLs and Overexpression of IGF-1R on the Release of Cytochrome c
To examine the role of cytochrome C in oxLDL-induced apoptosis, we incubated HASMCs with oxLDLs for various lengths of times. We found that oxLDLs increased cytosolic cytochrome C levels (Figure 4A) at 16 hours but not at 7 hours. In addition, the cells were exposed to oxLDLs for 7 or 16 hours, the oxLDLs were removed, new culture media were added, and cytochrome C release was determined 24 hours after the initial exposure to oxLDLs. The cytochrome C release induced after 16 hours was still present at 24 hours despite removal of oxLDLs (not shown). nLDLs did not affect cytosolic cytochrome C levels.

View larger version (27K): [in this window] [in a new window]   Figure 4. Effects of oxLDL, IGF-1, and overexpression of IGF-1R on cytosolic cytochrome C levels. Cytosolic cytochrome C levels were assayed by ELISA as described in the Materials and Methods. The results were expressed as a percentage of the control ± SEM. A, HASMCs were exposed to nLDLs (60 µg/mL) or oxLDLs (60 µg/mL) for 2.5, 7, or 16 hours. *P<0.01 compared with control, ¶P<0.01 compared with nLDL. B, HASMCs were exposed to serum-free medium alone (control) or with 60 µg/mL oxLDL in the absence or presence of IGF-1: IGF(2), IGF-1, 2 ng/mL; IGF(10) IGF-1, 10 ng/mL; IGF(50), IGF-1 50 ng/mL. ¶P<0.0025 compared with control, *P<0.025 compared with oxLDL, **P<0.005 compared with oxLDL. C, HASMCs were incubated alone (control) or with 60 µg/mL oxLDL for 24 hours or were infected with AdGFP or AdIGF-1R for 24 hours and then incubated with or without oxLDL (60 µg/mL) for 16 hours. *P<0.01 compared with control, ¶P<0.01 compared with AdGFP.

IGF-1 partially attenuated this oxLDL-induced increase in cytosolic cytochrome C levels (Figure 4B). However, IGF-1R overexpression using AdIGF-1R totally blocked the oxLDL-induced cytosolic cytochrome C release (Figure 4C).

Mechanisms Underlying the Ability of IGF-1R to Rescue Cells from oxLDL Cytotoxicity
To delineate the signal transduction pathways involved in the ability of the IGF-1R to rescue cells from oxLDL-induced apoptosis, we investigated the dual PI3 kinase/Akt and MAPK pathways. We found that infection of VSMCs with an adenovirus encoding the full-length IGF-1R increased the expression of IGF-1R (Figure 5A), the expression of phosphor-Akt and phosphor-Bad (Figure 5A), and phosphor-Erk42/44 (not shown). However, the overexpression of IGF-1R did not alter the expression of total Akt (Figure 5A) and total Erk42/44 (not shown). Moreover, the overexpression of GFP did not change the expression of IGF-1R, phosphor-Erk42/44, and phosphor-Akt. In vitro Akt kinase assays show that overexpression of the IGF-1R increased the activity of Akt kinase (Figure 5B). The dominant-negative PI3 kinase construct and the PI3 kinase inhibitor LY294002 blocked the ability of overexpressed IGF-1R to increase Akt kinase activity (Figure 5B). Neither the MAPKK (MEK) inhibitor PD98059 nor the control virus AdGFP altered the ability of AdIGF-1R to increase Akt kinase activity.

View larger version (35K): [in this window] [in a new window]   Figure 5. Effect of AdIGF-1R on the expression of IGF-1R, phosphor-Akt, and phosphor-Bad. A, HASMCs were infected with AdIGF-1R or control AdGFP for 24 hours and cell lysates subjected to 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and blotted with anti-IGF-1Rß, anti-actin, anti-total Akt, anti-pAkt, anti-total Bad, or anti-pBad antibody. Detection was by enhanced chemiluminescence. B, Effect of AdIGF-1R on the activity of Akt kinase. HASMCs were incubated alone (control) or infected with AdIGF-1R or AdGFP for 24 hours or treated with PI3 kinase inhibitor LY294002 (50 µmol/L) or MAPKK inhibitor PD98059 (50 µmol/L) in addition to being infected with AdIGF-1R for 24 hours or infected with AdIGF-1R and a dominant-negative PI3 kinase adenovirus (AdDN85). After Akt immunoprecipitation, an Akt kinase assay was conducted using GSK-3 fusion protein as substrate, and Western blotting was used to determine Akt kinase activity by measuring the level of phosphorylation of GSK-3 fusion protein with anti-phospho-GSK3 /ß antibody.

The PI3 kinase inhibitor LY294002 dose dependently blocked the ability of the overexpressed IGF-1R to rescue cells from oxLDL-induced apoptosis (Figure 6A). Moreover, infection with a dominant-negative PI3 kinase adenovirus (AdDN85) also inhibited the ability of the IGF-1R to block oxLDL-induced apoptosis (Figure 6B). In contrast, the MEK inhibitor PD98059 did not block IGF-1R–induced rescue from oxLDL-triggered apoptosis (not shown). These data indicate that the ability of the overexpressed IGF-1R to prevent oxLDL-induced loss of mitochondrial membrane potential, cytochrome C release, and apoptosis is mediated by the PI3 kinase/Akt pathway.

View larger version (29K): [in this window] [in a new window]   Figure 6. The PI3 kinase pathway mediates IGF-1R–induced rescue from oxLDL-triggered cell death. A, HASMCs were treated with 0 to 50 µmol/L PI3 kinase inhibitor LY294002 with or without AdIGF-1R for 24 hours and then exposed to oxLDL (0 to 60 µg/mL) for another 24 hours before cell viability was measured. Results are expressed as a percentage of the control ± SEM. ¶P<0.0025 compared with control (absence of oxLDL); *P<0.025 compared with AdIGF-1R + oxLDL; **P<0.0025 compared with AdIGF-1R+oxLDL. B, HASMCs were incubated alone (control) or with 60 µg/mL oxLDL for 24 hours or were infected with AdIGF-1R or AdDN85 or their combination for 24 hours and then exposed to oxLDL (0 to 60 µg/mL) for another 24 hours. Cell viability was determined and expressed as a percentage of the control ± SEM. *P<0.01 compared with control; **P<0.05 compared with AdDN85; ¶P<0.025 compared with AdDN85 + AdGFP; #P<0.005 compared with AdIGF-1R + oxLDL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of this study is that robust activation of the IGF-1R signaling pathway (and specifically of PI3-K/Akt) via overexpression of the IGF-1R completely rescues VSMCs from the apoptotic effects of oxLDLs. This finding has major implications for understanding the mechanisms of VSMC loss in atherosclerotic plaques because we have shown recently that oxLDLs reduce IGF-1 and IGF-1R expression in VSMCs in vitro⁹ and that areas of atherosclerotic plaque associated with expression of oxLDLs and increased VSMC apoptotic rates have lower expression of IGF-1/IGF-1R.^8,10 Indeed, Patel et al³² have shown that VSMCs from atherosclerotic plaque have reduced IGF-1R and reduced survival in vitro. Our findings may also be relevant to mechanisms of endothelial cell and macrophage damage in atherosclerotic lesions because both these cells may undergo apoptosis and/or nonapoptotic cell death in response to oxLDLs.^28,29,33 The other major finding of our study is that oxLDLs rapidly reduced mitochondrial membrane potential, resulting in cytosolic release of cytochrome c, and that this mechanism is critical for the apoptotic effect of oxLDLs on VSMCs and is completely inhibited by IGF-1R overexpression.

Our data showed that 7-hour of exposure to oxLDLs decreased without affecting cytochrome C release or apoptosis. Thus, the decrease in occurred before the release of cytochrome C (at 16 hours), suggesting that cytochrome C release induced by oxLDLs was a consequence of mitochondrial permeability transition pore opening. Our findings are consistent with reports that mitochondrial impairment leads to Apaf-1 activation of caspase networks, leading to apoptosis.³⁴ Moreover, the finding that only oxLDLs (and not nLDLs) could induce cytochrome C release and the fact that cytochrome C release (at 16 hours) occurred before toxicity was induced (at 24 hours) indicate that oxLDL-induced toxicity requires the release of cytochrome C from mitochondria, which is consistent with previous findings that cytochrome C release is necessary for apoptosis in many cell lines.^23,35

The IGF-1R signaling pathway has been shown to be a potent survival signal for a variety of cell types in vitro.^11,12,36,37 Depending on the cell type and the apoptotic stimuli, the survival effects can be mediated through the MAPK and/or protein kinase B (PKB)/Akt pathways.^13,36 In addition, novel pathways may be involved.^38–40 Mechanisms whereby oxLDLs can induce apoptosis of VSMCs are incompletely understood but may include changes in stimulation of tumor necrosis factor receptors⁴¹ and Fas,^41,42 MAP, and Jun kinases,⁴³ and the generation of reactive oxygen species.⁴⁴ Our previous report that oxLDLs downregulate IGF-1/IGF-1R expression in rat aortic VSMCs⁹ (which we have reproduced using HASMCs; P. Delafontaine, unpublished results, 2003) suggested that downregulation of this autocrine pathway could play a pivotal role in oxLDL-induced apoptosis of VSMCs. Indeed, our present findings demonstrating the ability of IGF-1R overexpression to rescue VSMCs from oxLDL-induced apoptosis suggest strongly that reduced signaling through this pathway plays an important role in oxLDL apoptotic effects. Furthermore, the inability of excess exogenous IGF-1 (or des-IGF-1) to fully rescue cells from oxLDL apoptosis suggests that IGF-1R density is a critical regulator of IGF-1-mediated survival effects, analogous to the critical role of IGF-1R density in IGF-1 mitogenic responses in VSMCs^45–47 and other cell types.^48–51 Furthermore, the ability of IGF-1R overexpression to prevent an oxLDL-induced decrease in mitochondrial membrane potential and cytosolic cytochrome C release demonstrates that the overexpression of this receptor results in the inhibition of a mechanism that is central to both stress-induced and death receptor–induced apoptosis^25,26 and that participates in nonapoptotic (oncotic) cell death.²⁹ Furthermore, these findings establish a central role for mitochondrial dysfunction in the apoptotic effects of oxLDLs on VSMCs, similarly to a recent report on oxLDL’s effects on endothelial cells.²⁸

The present study examined the signaling pathways mediating the ability of the overexpressed IGF-IR to rescue VSMCs from oxLDL-induced apoptosis and documented that this survival pathway signals through PI3 kinase and Akt based on the following lines of evidence: (1) PI3 kinase inhibition abolished the ability of the overexpressed IGF-1R to prevent oxLDL-induced cytotoxicity; (2) overexpression of a dominant-negative PI3 kinase construct using AdDN85 abolished the capacity of the IGF-1R to preserve VSMC viability under oxLDL exposure; and (3) overexpression of IGF-1R increased the kinase activity of Akt and PI3 kinase inhibition and a dominant-negative AdDN85 blocked IGF-1R–induced Akt kinase activity. Although overexpression of IGF-1R increased the expression of phosphor-Erk42/44, the MEK inhibitor PD98059 (which acts upstream of Erk42/44) did not block IGF-1R–induced survival effects and the increase in Akt kinase activity. Taken together, these findings provide substantial evidence that PI3 kinase/Akt rather than MAPK is the dominant signaling pathway underlying IGF-1R–mediated inhibition of oxLDL-induced apoptosis of VSMCs. The marked increase in Akt phosphorylation and activity in response to IGF-1R overexpression would be expected to be a potent survival signal through the known ability of activated Akt to intervene in the apoptotic cascade upstream of the mitochondria, namely through its phosphorylation and inactivation of proapoptotic proteins Bad and potentially others.^52,53 Our data that IGF-1R–triggered activation of Akt results in inhibition of oxLDL-induced changes in mitochondrial membrane potential and cytosolic cytochrome C release are consistent with the report that activated Akt can prevent UV light–mediated mitochondrial permeability change, cytosolic cytochrome C release, and cell death.²²

In view of our recent report demonstrating reduced IGF-1 and IGF-1R expression in atherosclerotic plaque in addition to the report by Patel et al³² demonstrating reduced IGF-1R and reduced survival of VSMCs from atherosclerotic plaque, these findings have important implications for understanding mechanisms of VSMC depletion that could potentially contribute to plaque destabilization. Thus, there have been multiple reports that unstable plaques have a relative depletion of VSMCs, particularly in areas with a high number of inflammatory cells, such as the shoulder regions.^54–57 Furthermore, unstable plaques are characterized by thin fibrous caps, again consistent with a relative paucity of VSMCs.^54–57 The ability of oxLDLs to decrease IGF-1 and IGF-1R expression and, more importantly, the ability of IGF-1R overexpression to completely rescue VSMCs from oxLDL-induced cytotoxicity suggest that manipulation of the IGF-1/IGF-1R autocrine system may offer an attractive strategy for plaque stabilization.

In summary, overexpression of the IGF-1R in VSMCs inhibits oxLDL-induced membrane permeabilization and cytochrome C release and rescues these cells from oxLDL-induced apoptosis through a PI3 kinase/Akt–dependent signaling pathway. These findings suggest that manipulation of the IGF-1/IGF-1R autocrine/paracrine pathway may be a useful strategy to limit the loss of VSMCs that contribute to atherosclerotic plaque destabilization.

	Acknowledgments

 
Acknowledgments

This study was supported by National Institutes of Health Grant 1 RO1 HL 70241–01A1 (to P.D.), National Institutes of Health Grant 1 RO1 HL70762–01 (to J.D.), and American Heart Association National Grant 0030163N (to J.D.). The authors thank Laura Blalock for editorial assistance.

Received September 3, 2003; accepted September 25, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Bulter S, Wittztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989; 84: 1086–1095.[Medline] [Order article via Infotrieve]

2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989; 320: 915–924.[Medline] [Order article via Infotrieve]

3. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991; 88: 1785–1792.[Medline] [Order article via Infotrieve]

4. Juckett MB, Balla J, Balla G, Jessurun J, Jacob HS, Vercellotti GM. Ferritin protects endothelial cells from oxidized low density lipoprotein in vitro. Am J Pathol. 1995; 147: 782–789.[Abstract]

5. Dimmeler S, Haendeler J, Galle J, Zeiher AM. Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases: a mechanistic clue to the ’response to injury’ hypothesis. Circulation. 1997; 95: 1760–1763.[Abstract/Free Full Text]

6. Nishio E, Watanabe Y. Oxysterols induced apoptosis in cultured smooth muscle cells through CPP32 protease activation and bcl-2 protein downregulation. Biochem Biophys Res Commun. 1996; 226: 928–934.[CrossRef][Medline] [Order article via Infotrieve]

7. Siow RC, Richards JP, Pedley KC, Leake DS, Mann GE. Vitamin C protects human vascular smooth muscle cells against apoptosis induced by moderately oxidized LDL containing high levels of lipid hydroperoxides. Arterioscler Thromb Vasc Biol. 1999; 19: 2387–2394.[Abstract/Free Full Text]

8. Okura Y, Brink M, Itabe H, Scheidegger KJ, Kalangos A, Delafontaine P. Oxidized low-density lipoprotein is associated with apoptosis of vascular smooth muscle cells in human atherosclerotic plaques. Circulation. 2000; 102: 2680–2686.[Abstract/Free Full Text]

9. Scheidegger KJ, James RW, Delafontaine P. Differential effects of low density lipoproteins on insulin-like growth factor-1 (IGF-1) and IGF-1 receptor expression in vascular smooth muscle cells. J Biol Chem. 2000; 275: 26864–26869.[Abstract/Free Full Text]

10. Okura Y, Brink M, Zahid AA, Anwar A, Delafontaine P. Decreased expression of insulin-like growth factor-1 and apoptosis of vascular smooth muscle cells in human atherosclerotic plaque. J Mol Cell Cardiol. 2001; 33: 1777–1789.[CrossRef][Medline] [Order article via Infotrieve]

11. Zheng WH, Kar S, Quirion R. Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells. J Biol Chem. 2000; 275: 39152–39158.[Abstract/Free Full Text]

12. Dore S, Kar S, Quirion R. Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity. Proc Natl Acad Sci U S A. 1997; 94: 4772–4777.[Abstract/Free Full Text]

13. Parrizas M, Saltiel AR, LeRoith D. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem. 1997; 272: 154–161.[Abstract/Free Full Text]

14. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001; 414: 799–806.[CrossRef][Medline] [Order article via Infotrieve]

15. Ueki K, Fruman DA, Brachmann SM, Tseng YH, Cantley LC, Kahn CR. Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival. Mol Cell Biol. 2002; 22: 965–977.[Abstract/Free Full Text]

16. Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol. 1997; 17: 1595–1606.[Abstract/Free Full Text]

17. Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995; 267: 2003–2006.[Abstract/Free Full Text]

18. Dudek H, Datta SR, Franke TF, Yao R, Cooper GM, Segal RA, Kaplan DR, Greenberg ME. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science. 1997; 275: 661–665.[Abstract/Free Full Text]

19. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997; 278: 687–689.[Abstract/Free Full Text]

20. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998; 282: 1318–1321.[Abstract/Free Full Text]

21. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Luo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell. 1999; 96: 857–868.[CrossRef][Medline] [Order article via Infotrieve]

22. Kennedy SG, Kandel ES, Cross TK, Hay N. Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol Cell Biol. 1999; 19: 5800–5810.[Abstract/Free Full Text]

23. Reed JC. Cytochrome c: can’t live with it—can’t live without it. Cell. 1997; 91: 559–62.[CrossRef][Medline] [Order article via Infotrieve]

24. Zhivotovsky B, Orrenius S, Brustugun OT, Doskeland SO. Injected cytochrome c induces apoptosis. Nature. 1998; 391: 449–450.[Medline] [Order article via Infotrieve]

25. Kumar S, Vaux DL. Apoptosis: a Cinderella caspase takes center stage. Science. 2002; 297: 1290–1291.[Free Full Text]

26. Lassus P, Opitz-Araya X, Lazebnik Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science. 2002; 297: 1352–1354.[Abstract/Free Full Text]

27. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997; 91: 479–489.[CrossRef][Medline] [Order article via Infotrieve]

28. Walter DH, Haendeler J, Galle J, Zeiher AM, Dimmeler S. Cyclosporin A inhibits apoptosis of human endothelial cells by preventing release of cytochrome C from mitochondria. Circulation. 1998; 98: 1153–1157.[Abstract/Free Full Text]

29. Asmis R, Begley JG. Oxidized LDL promotes peroxide-mediated mitochondrial dysfunction and cell death in human macrophages: a caspase-3-independent pathway. Circ Res. 2003; 92: e20–29.[Abstract/Free Full Text]

30. Jacob T, Hingorani A, Ascher E. Examination of the apoptotic pathway and proteolysis in the pathogenesis of popliteal artery aneurysms. Eur J Vasc Endovasc Surg. 2001; 22: 77–85.[CrossRef][Medline] [Order article via Infotrieve]

31. Sakaki T, Kohmura E, Kishiguchi T, Yuguchi T, Yamashita T, Hayakawa T. Loss and apoptosis of smooth muscle cells in intracranial aneurysms: studies with in situ DNA end labeling and antibody against single-stranded DNA. Acta Neurochir (Wien). 1997; 139: 469–74;discussion 474–475.

32. Patel VA, Zhang QJ, Siddle K, Soos MA, Goddard M, Weissberg PL, Bennett MR. Defect in insulin-like growth factor-1 survival mechanism in atherosclerotic plaque-derived vascular smooth muscle cells is mediated by reduced surface binding and signaling. Circ Res. 2001; 88: 895–902.[Abstract/Free Full Text]

33. Heinloth A, Brune B, Fischer B, Galle J. Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation. Atherosclerosis. 2002; 162: 93–101.[CrossRef][Medline] [Order article via Infotrieve]

34. Shidoji Y, Nakamura N, Moriwaki H, Muto Y. Rapid loss in the mitochondrial membrane potential during geranylgeranoic acid-induced apoptosis. Biochem Biophys Res Commun. 1997; 230: 58–63.[CrossRef][Medline] [Order article via Infotrieve]

35. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, jones DP, Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997; 275: 1129–1132.[Abstract/Free Full Text]

36. Zheng WH, Kar S, Quirion R. Insulin-like growth factor-1-induced phosphorylation of transcription factor FKHRL1 is mediated by phosphatidylinositol 3-kinase/Akt kinase and role of this pathway in insulin-like growth factor-1-induced survival of cultured hippocampal neurons. Mol Pharmacol. 2002; 62: 225–233.[Abstract/Free Full Text]

37. Kiely PA, Sant A, O’Connor R. RACK1 is an insulin-like growth factor 1 (IGF-1) receptor-interacting protein that can regulate IGF-1-mediated Akt activation and protection from cell death. J Biol Chem. 2002; 277: 22581–22589.[Abstract/Free Full Text]

38. Linseman DA, Phelps RA, Bouchard RJ, Le SS, Laessig TA, McClure ML, Heidenreich KA. Insulin-like growth factor-I blocks Bcl-2 interacting mediator of cell death (Bim) induction and intrinsic death signaling in cerebellar granule neurons. J Neurosci. 2002; 22: 9287–9297.[Abstract/Free Full Text]

39. Walsh PT, Smith LM, O’Connor R. Insulin-like growth factor-1 activates Akt and Jun N-terminal kinases (JNKs) in promoting the survival of T lymphocytes. Immunology. 2002; 107: 461–471.[CrossRef][Medline] [Order article via Infotrieve]

40. Bonthius DJ, Karacay B, Dai D, Pantazis NJ. FGF-2, NGF and IGF-1, but not BDNF, utilize a nitric oxide pathway to signal neurotrophic and neuroprotective effects against alcohol toxicity in cerebellar granule cell cultures. Brain Res Dev Brain Res. 2003; 140: 15–28.[CrossRef][Medline] [Order article via Infotrieve]

41. Napoli C, Quehenberger O, De Nigris F, Abete P, Glass CK, Palinski W. Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells. Faseb J. 2000; 14: 1996–2007.[Abstract/Free Full Text]

42. Lee T, Chau L. Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol. 2001; 280: C709–718.[Abstract/Free Full Text]

43. Jing Q, Xin SM, Cheng ZJ, Zhang WB, Zhang R, Qin YW, Pei G. Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells: mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity. Circ Res. 1999; 84: 831–839.[Abstract/Free Full Text]

44. Hsieh CC, Yen MH, Yen CH, Lau YT. Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells. Cardiovasc Res. 2001; 49: 135–145.[Abstract/Free Full Text]

45. Du J, Delafontaine P. Inhibition of vascular smooth muscle cell growth through antisense transcription of a rat insulin-like growth factor I receptor cDNA. Circ Res. 1995; 76: 963–972.[Abstract/Free Full Text]

46. Delafontaine P, Meng XP, Ku L, Du J. Regulation of vascular smooth muscle cell insulin-like growth factor I receptors by phosphorothioate oligonucleotides. Effects on cell growth and evidence that sense targeting at the ATG site increases receptor expression. J Biol Chem. 1995; 270: 14383–14388.[Abstract/Free Full Text]

47. Delafontaine P, Anwar A, Lou H, Ku L. G-protein coupled and tyrosine kinase receptors: evidence that activation of the insulin-like growth factor I receptor is required for thrombin-induced mitogenesis of rat aortic smooth muscle cells. J Clin Invest. 1996; 97: 139–145.[Medline] [Order article via Infotrieve]

48. Pass HI, Mew DJ, Carbone M, Donington JS, Baserga R, Steinberg SM. The effect of an antisense expression plasmid to the IGF-1 receptor on hamster mesothelioma proliferation. Dev Biol Stand. 1998; 94: 321–328.[Medline] [Order article via Infotrieve]

49. Swantek JL, Baserga R. Prolonged activation of ERK2 by epidermal growth factor and other growth factors requires a functional insulin-like growth factor 1 receptor. Endocrinology. 1999; 140: 3163–3169.[Abstract/Free Full Text]

50. Steller MA, Zou Z, Schiller JT, Baserga R. Transformation by human papillomavirus 16 E6 and E7: role of the insulin-like growth factor 1 receptor. Cancer Res. 1996; 56: 5087–5091.[Abstract/Free Full Text]

51. Pass HI, Mew DJ, Carbone M, Donington JS, Baserga R, Steinberg SM. Inhibition of hamster mesothelioma tumorigenesis by an antisense expression plasmid to the insulin-like growth factor-1 receptor. Cancer Res. 1996; 56: 4044–4048.[Abstract/Free Full Text]

52. Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002; 296: 1655–1657.[Abstract/Free Full Text]

53. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999; 13: 2905–2927.[Free Full Text]

54. Libby P. Coronary artery injury and the biology of atherosclerosis: inflammation, thrombosis, and stabilization. Am J Cardiol. 2000; 86: 3J–8J;discussion 8J–9J.

55. Martinet W, Kockx MM. Apoptosis in atherosclerosis: focus on oxidized lipids and inflammation. Curr Opin Lipidol. 2001; 12: 535–541.[CrossRef][Medline] [Order article via Infotrieve]

56. Kolodgie FD, Narula J, Haider N, Virmani R. Apoptosis in atherosclerosis. Does it contribute to plaque instability? Cardiol Clin. 2001; 19: 127–139, ix.[CrossRef][Medline] [Order article via Infotrieve]

57. Burke AP, Kolodgie FD, Farb A, Weber D, Virmani R. Morphological predictors of arterial remodeling in coronary atherosclerosis. Circulation. 2002; 105: 297–303.[Abstract/Free Full Text]

This article has been cited by other articles:

				J. S. Titterington, S. Sukhanov, Y. Higashi, C. Vaughn, C. Bowers, and P. Delafontaine Growth Hormone-Releasing Peptide-2 Suppresses Vascular Oxidative Stress in ApoE-/- Mice But Does Not Reduce Atherosclerosis Endocrinology, December 1, 2009; 150(12): 5478 - 5487. [Abstract] [Full Text] [PDF]

				T. W. Small and J. G. Pickering Nuclear Degradation of Wilms Tumor 1-associating Protein and Survivin Splice Variant Switching Underlie IGF-1-mediated Survival J. Biol. Chem., September 11, 2009; 284(37): 24684 - 24695. [Abstract] [Full Text] [PDF]

				Y. Higashi, S. Sukhanov, S. Parthasarathy, and P. Delafontaine The ubiquitin ligase Nedd4 mediates oxidized low-density lipoprotein-induced downregulation of insulin-like growth factor-1 receptor Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1684 - H1689. [Abstract] [Full Text] [PDF]

				D. Allard, N. Figg, M. R. Bennett, and T. D. Littlewood Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3 J. Biol. Chem., July 11, 2008; 283(28): 19739 - 19747. [Abstract] [Full Text] [PDF]

				R. M. Martin, D. Gunnell, E. Whitley, A. Nicolaides, M. Griffin, N. Georgiou, G. Davey Smith, S. Ebrahim, and J. M. P. Holly Associations of Insulin-Like Growth Factor (IGF)-I, IGF-II, IGF Binding Protein (IGFBP)-2 and IGFBP-3 with Ultrasound Measures of Atherosclerosis and Plaque Stability in an Older Adult Population J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1331 - 1338. [Abstract] [Full Text] [PDF]

				S. Sukhanov, Y. Higashi, S.-Y. Shai, C. Vaughn, J. Mohler, Y. Li, Y.-H. Song, J. Titterington, and P. Delafontaine IGF-1 Reduces Inflammatory Responses, Suppresses Oxidative Stress, and Decreases Atherosclerosis Progression in ApoE-Deficient Mice Arterioscler Thromb Vasc Biol, December 1, 2007; 27(12): 2684 - 2690. [Abstract] [Full Text] [PDF]

				S. Sukhanov, Y. Higashi, S.-Y. Shai, H. Itabe, K. Ono, S. Parthasarathy, and P. Delafontaine Novel Effect of Oxidized Low-Density Lipoprotein: Cellular ATP Depletion via Downregulation of Glyceraldehyde-3-Phosphate Dehydrogenase Circ. Res., July 21, 2006; 99(2): 191 - 200. [Abstract] [Full Text] [PDF]

				Y. Li, Y.-H. Song, J. Mohler, and P. Delafontaine ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H2116 - H2123. [Abstract] [Full Text] [PDF]

				Y. Higashi, T. Peng, J. Du, S. Sukhanov, Y. Li, H. Itabe, S. Parthasarathy, and P. Delafontaine A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor J. Lipid Res., June 1, 2005; 46(6): 1266 - 1277. [Abstract] [Full Text] [PDF]

				P. Delafontaine, Y.-H. Song, and Y. Li Expression, Regulation, and Function of IGF-1, IGF-1R, and IGF-1 Binding Proteins in Blood Vessels Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 435 - 444. [Abstract] [Full Text]

				N. Kume and T. Kita Apoptosis of Vascular Cells by Oxidized LDL: Involvement of Caspases and LOX-1 and Its Implication in Atherosclerotic Plaque Rupture Circ. Res., February 20, 2004; 94(3): 269 - 270. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 23/12/2178    most recent 01.ATV.0000099788.31333.DBv1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, Y. Articles by Delafontaine, P. Search for Related Content PubMed PubMed Citation Articles by Li, Y. Articles by Delafontaine, P. Related Collections Apoptosis Pathophysiology Growth factors/cytokines Mechanism of atherosclerosis/growth factors