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(Circulation Research. 2001;0:hh0901.090305.)
© 2001 American Heart Association, Inc.

Article

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

V. A. Patel, Q.-J. Zhang, K. Siddle, M. A. Soos, M. Goddard, P. L. Weissberg M. R. Bennett

From the Departments of Medicine (V.A.P., Q.-J.Z., M.A.S., P.L.W., M.R.B.) and Clinical Biochemistry (K.S., M.A.S.), Addenbrooke’s Hospital, Cambridge, UK; and Department of Histopathology (M.G.), Papworth Hospital, Cambridge, UK.

Correspondence to Prof Martin Bennett, Division of Cardiovascular Medicine, Addenbrooke’s Centre for Clinical Investigation, Box 110, Addenbrooke’s Hospital, Cambridge Hills Rd, Cambridge CB2 2QQ, UK. E-mail to mrb{at}mole.bio.cam.ac.uk

Abstract

Abstract—Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque–derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% ¹²⁵I–IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase–mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1–mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.

Key Words: apoptosis • atherosclerosis • Akt • plaque rupture • insulin-like growth factor-1 signaling

The precipitating event in coronary artery occlusion leading to myocardial infarction is usually the rupture of the atherosclerotic plaque, often at a weak point in the shoulder region of the fibrous cap, with subsequent thrombosis over the plaque. Thus, any change in plaque composition, which destabilizes the vascular smooth muscle cell (VSMC)-rich cap is potentially dangerous, such as a reduction in VSMC numbers, or breakdown of extracellular matrix.^{1 2 3} It has been recognized for many years that (necrotic) cell death reduces VSMC numbers in advanced atherosclerosis, in both the fibrous cap and the lipid core region (reviewed by Schwartz and Bennett⁴ ). However, the recent observation that apoptosis occurs in advanced plaques,^{5 6} including at the shoulder regions, indicates that specific gene products and local cytokines, including survival factors, may regulate cell loss in atherosclerosis.>

Insulin-like growth factor (IGF-1) is a polypeptide growth factor that binds to the specific type I IGF-1 receptor (IGF-1R) present on many cell types, including VSMCs.⁷ IGF-1 is a weak mitogen for VSMCs,^{8 9 10} but it is a potent survival factor for many cell types, including fibroblasts, VSMCs, neurons, cardiac myocytes, and tumor cells.^{11 12 13 14 15} Indeed, the suppression of IGF-1 signaling induces massive apoptosis in vitro and in vivo,¹⁶ and the administration of IGF-1 can suppress apoptosis in vivo, for example, in cardiac myocytes after ischemia and reperfusion.¹⁷ >

Although the precise pathways mediating the survival action of IGF-1 are unclear, the IGF-1R possesses intrinsic tyrosine kinase activity and activates a number of downstream mediators, including insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI 3-kinase), and mitogen-activated protein kinase (MAPK). PI 3-kinase is antiapoptotic in many cell types,¹⁸ and some of the survival function of IGF-1 is mediated by PI 3-kinase.^{19 20 21} In addition, IGF-1 promotes cell proliferation by activation of the p42 ERK (MAPK) pathway, resulting in phosphorylation of both extra cellular–signal related kinase (ERK)-1 and ERK-2, which may promote or protect against apoptosis, depending on the system studied.^{19 20 22}

Recently, downstream targets that mediate IGF-1 protection have emerged. Activation of PI 3-kinase leads to activation of the serine/threonine kinase Akt, which itself phosphorylates the proapoptotic proteins Bad, caspase 9, and FKHRL1, a Forkhead family transcription factor.^{23 24 25} Bad is a BCl-2 family gene that induces apoptosis by interaction with other BCl-2 family members. Phosphorylation of Bad renders this protein inactive, preventing this association.²³ Phosphorylation of FKHRL1 also sequesters it in the cytoplasm, inhibiting nuclear translocation and transcription of target genes. In contrast, caspase 9 is a cysteine protease intimately involved with regulating and executing apoptosis. Caspase 9 cleavage occurs as part of the formation of a proapoptotic complex, the apoptosome, which is responsible for signaling apoptosis from mitochondria. Akt phosphorylation of caspase 9 prevents its cleavage and activation, thus inhibiting apoptosis.²⁴

Preliminary evidence suggested defective survival signaling through IGF-1 in human atherosclerotic plaque VSMCs (pVSMCs). IGF-1 replacement totally rescued growth factor withdrawal–induced apoptosis of normal VSMCs (nVSMC), but not pVSMCs.²⁶ In addition, some reports indicated that the tumor suppressor gene p53 may induce apoptosis in part by inducing expression of the IGF-1 binding protein (IGFBP)-3,²⁷ and/or by directly inhibiting transcription of IGF-1R.²⁸ p53 selectively induces plaque but not nVSMC apoptosis.^{29 30} We therefore examined the regulation of human pVSMC apoptosis by IGF-1.>

Materials and Methods

Cell Culture
VSMCs from human atherosclerotic plaques were derived from carotid endarterectomies and normal medial VSMCs from aortas of patients undergoing renal or hepatic transplantation at Addenbrooke’s Hospital, approved by the Local Ethics Committee. Cells were cultured in M199 medium containing 20% FCS. The generation of semi-immortalized human VSMCs expressing either simian virus 40 (SV40) large T antigen or human papilloma virus E6 (HPV E6) has been described before.³¹

Time-Lapse Videomicroscopy
Cells were prepared for videomicroscopy as previously described.³¹

Radioligand Binding
Confluent cells (65%) were incubated in ligand binding-buffer containing 4x10³ cpm ¹²⁵I–IGF-1 with or without excess cold IGF-1 (100 nmol/L) or with or without cold insulin (10 µmol/L) at 4°C for 6 hours with shaking. Cell layers were washed, solubilized, and counted on a gamma counter. Duplicate plates were used to determine cell number to calculate binding per cell.

Akt Kinase Assay
Akt kinase activity was assayed by using a glycogen synthase kinase-3–based peptide (GSK-3) (Upstate Biotechnology). Equal amounts of protein were loaded and electrophoresed before blotting or were immunoprecipitated to incorporate radiolabel into GSK-3.

Western Blotting
Immunoblot analysis was used to determine IGF-1R and Akt activity by using phosphospecific antibodies in cell lysates and IGFBP levels in conditioned media (CM).

Immunocytochemistry
Immunocytochemistry was performed for IGF-1R, CD68, and actin on human coronary arteries by using standard methods.³²

Statistical Analysis
Statistical analysis was performed by using ANOVA and Student’s t test where appropriate.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

Results

IGF-1 Fails to Protect Plaque VSMCs From Apoptosis
We first examined the ability of IGF-1 to protect nVSMCs and pVSMCs from apoptosis after growth factor withdrawal. Asynchronous cells were transferred to serum-free medium (SFM) with or without IGF-1, and apoptosis was monitored by time-lapse videomicroscopy. IGF-1, 50 ng/mL, reduced nVSMC apoptosis by 50%. In contrast, SFM induced higher levels of pVSMC apoptosis that was not protected by 50 ng/mL IGF-1 (Figure 1A). Increasing IGF-1 concentrations (to 100 ng/mL) increased nVSMC survival. In contrast, there was a small survival effect of IGF-1 to 5 ng/mL in pVSMCs, but further increases did not increase protection (Figure 1B), indicating that pVSMCs are refractory to IGF-1–mediated survival. Of note, apoptosis was a stochastic phenomenon in VSMCs, uniform within the cultures, rather than occurring in any one subset. Extension of the experimental period to 72 hours showed that differences between nVSMC and pVSMC apoptotic rates were maintained (not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of IGF-1 on pVSMC and nVSMC survival. A, nVSMCs and pVSMCs were placed in 0% FCS medium with 50 ng/mL IGF-1, and cell death was recorded to 24 hours. n=4; *P<0.05 at 24 hours vs 0% FCS. B, XTT cell viability assay at 24 hours for VSMCs treated with increasing concentrations of IGF-1 in serum-free conditions. n=10; *P<0.05 vs the previous data point.

Plaque VSMCs Show Reduced Surface IGF-1 Binding and Type I Receptor Expression
To examine the mechanism of reduced IGF-1 survival in pVSMCs, we first performed radioligand-binding assays by using ¹²⁵I-labeled IGF-1. As IGF-1 binds both type I and II IGF receptors and IGFBPs, specific binding of IGF-1 to type I receptors was examined by using competition with insulin, which binds with low affinity to type I receptors, but not at all to type II receptors and IGFBPs. pVSMCs and nVSMCs showed similar levels of ¹²⁵I–IGF-1 binding (Figure 2A). However, most of this binding was not competed by high concentrations of unlabeled insulin. The component of ¹²⁵I–IGF-1–binding that was competed by insulin, which reflects type I receptors, was markedly reduced in pVSMCs versus nVSMCs (Figure 2B) (5.6±3 versus 25.7±11.1 arbitrary units [mean±SEM], n=3, P<0.05). The type I receptor–attributable specific binding to pVSMC or nVSMC cell lines was similar to untransformed pVAMCs or nVSMCs (not shown).

View larger version (24K): [in this window] [in a new window]   Figure 2. IGF-1 radioligand binding and IGF-1R expression in VSMCs. A, Radioligand binding assay for 2 pVSMC isolates and 1 nVSMC isolate with or without excess insulin (10 µmol/L). Cells in the monolayer were incubated with 125I–IGF-1, and bound cpm/5x104 cells were determined. n=4; *P<0.05. Nonspecific binding in the presence of 100 nmol/L cold IGF-1 has been subtracted. B, Specific binding to IGF-1R as measured by the difference in binding with or without excess insulin in 4 pVSMC and 4 nVSMC isolates. C, Western blot analysis of total expression of IGF-1R in 6 pVSMC and 3 nVSMC isolates and -actin loading control.

As most ¹²⁵I–IGF-1 binding to cells appeared to be to type II IGF receptors or cell-associated IGFBPs, it was not possible to perform a meaningful Scatchard analysis of IGF binding specifically to type I receptors to determine whether decreased binding to pVSMCs was caused by reduced receptor expression or reduced binding affinity. We therefore examined expression of type I IGF receptors by Western blotting with a specific antibody. pVSMCs showed markedly reduced expression of type I IGF-1R compared with nVSMCs (Figure 2C) (ratio of plaque and normal IGF-1R signal versus actin signal arbitrary units by densitometry, plaque 0.16±0.02 versus normal 0.94±0.03, n=9, P<0.001). VSMC lines showed increased expression of IGF-1R compared with untransformed cells, consistent with p53 suppression of IGF-1R expression. However, pVSMC lines maintained reduced expression compared with their normal medial counterparts (not shown). Reverse transcriptase–polymerase chain reaction (RT-PCR) for IGF-1R demonstrated that the reduced IGF-1R protein expression in pVSMCs was associated with reduced IGF-1R mRNA expression (not shown).

Plaque VSMCs Show Reduced Akt Signaling in Response to IGF-1
The inability of IGF-1 to rescue pVSMCs from apoptosis and decreased expression of type I IGF receptors in pVSMCs suggest that pVSMCs have reduced survival signaling in response to IGF-1. We therefore examined Akt phosphorylation after exogenous IGF-1 in VSMCs. VSMC lines were used for these and subsequent experiments in view of the difficulty in maintaining long-term cultures of pVSMCs and the heterogeneity of these cultures. The addition of IGF-1 to serum-starved nVSMCs induced rapid and robust Akt phosphorylation (Figure 3A). In contrast, although Akt phosphorylation was observed in pVSMCs, this was reduced compared with nVSMCs. Total Akt expression did not change over the same time course.

View larger version (29K): [in this window] [in a new window]   Figure 3. Akt signaling in pVSMCs in response to IGF-1. A, Western blot analysis with a phosphospecific antibody for Akt(ser473) or total Akt in pVSMCs and nVSMCs stimulated with IGF-1 (50 ng/mL) for 0 to 60 minutes. B, In vitro kinase assay for Akt in nVSMCs and pVSMCs stimulated with IGF-1 (50 ng/mL) for 0, 5, and 10 minutes. n=3; *P<0.05.

To confirm that reduced and delayed Akt phosphorylation reduced Akt activity in pVSMCs, we examined Akt kinase activity after the addition of IGF-1 by in vitro kinase assays, using a peptide based on GSK-3, a known Akt substrate. Kinase assays confirmed both reduced and delayed Akt activity in pVSMCs versus nVSMCs in response to IGF-1 (Figure 3B) (13604±4905 versus 34861±9510 cpm incorporated, n=3, P<0.05).

Plaque VSMCs Are Protected Against Apoptosis by IGF-1 Analogues
Although pVSMCs showed reduced IGF-1R expression and Akt signaling, the relative resistance of pVSMCs to IGF-1–mediated survival may also reflect different expression of IGFBPs, inhibiting IGF-1 binding to IGF-1R. We therefore examined the protection afforded by two IGF-1 analogues, long R3 (LR3) and Des 1,3, which bind IGF-1R with the same affinity as IGF-1, but do not bind IGFBPs.^{33 34} LR3 and Des 1,3 were equipotent to IGF-1 in nVSMCs, protecting against apoptosis in SFM. However, in contrast to IGF-1, LR3 and Des 1,3 potently inhibited apoptosis in pVSMCs to levels seen in nVSMCs (Figures 4A and 4B). We next examined the mechanism of LR3-induced protection. Interestingly, LR3 induced Akt phosphorylation to a similar extent and with kinetics similar to IGF-1 in pVSMCs (Figure 4C), indicating that differential Akt signaling was not responsible for the increased potency of LR3. We next examined whether Akt phosphorylation after IGF-1 in pVSMCs was mediated through PI 3-kinase. The PI 3-kinase inhibitors LY294009 and wortmannin significantly reduced LR3-induced Akt phosphorylation in pVSMCs (Figure 4D).

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of IGF-1 analogues on pVSMC apoptosis. nVSMCs (A) and pVSMCs (B) were placed in 0% FCS medium with 50 ng/mL IGF-1, LR3, or Des 1,3, and cell death was recorded to 24 hours. n=4; *P<0.05 at 24 hours vs 0% FCS. C, Western blot analysis with a phosphospecific antibody of Akt(ser473) or total Akt in pVSMCs stimulated with IGF-1 or LR3 (50 ng/mL) for 0 to 60 minutes. D, Western blots of phosphorylated Akt in pVSMCs in response to LR3 in the presence of LY294009 (20 µmol/L) or wortmannin (200 nmol/L).

IGF-1 and LR3 Induce p42/44 MAPK in pVSMCs
The potent survival action of LR3 compared with IGF-1 in pVSMCs despite similar Akt phosphorylation suggests that other mediators are responsible for LR3-induced protection. IGF-1 can also protect against apoptosis by activation of raf/MAPK signaling, as well as through PI 3-kinase. We therefore examined phosphorylation of p42/44 MAPK after IGF-1 administration and the ability of the MAP/ERK kinase inhibitor PD98059 to block IGF-1–induced survival. Both IGF-1 and LR3 induced p42/44 MAPK phosphorylation in pVSMCs, with LR3 being more potent (Figure 5A). PD98059 also blocked p42/44 ERK-1 and -2 phosphorylation induced by LR3 (Figure 5B). However, unlike LY294009, PD98059 did not block LR3 protection of pVSMC apoptosis (Figure 5C).

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of ERK inhibition on LR3-induced pVSMC survival. A, Western blot for phosphorylated p42/44 MAPK after IGF-1 or LR3. pVSMCs were placed in low-serum medium in the presence of IGF-1 or LR3 for 0 to 60 minutes. B, Western blot for phosphorylated p42/44 MAPK after LR3 treatment at 10 minutes in the presence or absence of PD98059 (20 µmol/L). C, Cumulative cell death in pVSMCs. pVSMCs were placed in low-serum medium in the presence of LR3 (50 ng/mL) with the vehicle alone (DMSO), PD98059 (20 µmol/L), or LY294009 (20 µmol/L), and deaths were recorded to 24 hours. n=4; *P<0.05 at 24 hours vs DMSO control.

Plaque VSMCs Synthesize High Levels of IGFBPs
The ability of LR3 but not IGF-1 to inhibit apoptosis in pVSMCs suggests that pVSMCs synthesize and secrete IGFBPs, which inhibit IGF-1 binding to IGF-1R. We therefore examined IGFBP expression using RT-PCR. pVSMCs expressed increased IGFBP-2 to -6 mRNA (Figure 6A). IGFBP expression and secretion into CM was measured by using Western blotting. pVSMCs showed increased secretion of IGFBP-2, -3, and -4 in CM (Figure 6B) compared with nVSMCs. VSMCs transformed with SV40 or HPV E6 showed slightly reduced IGFBP secretion compared with primary cells, although differences in expression of IGFBPs between transformed pVSMCs versus nVSMCs were maintained (not shown). In addition, we examined the ability of the IGFBPs secreted by pVSMCs to block the protection of nVSMCs by IGF-1 and LR3. pVSMC-CM induced higher apoptosis in nVSMCs than nVSMC-CM. This effect could be partially reversed by IGF-1 and more fully reversed by LR3, indicating that IGFBPs were responsible (Figure 6C).

View larger version (29K): [in this window] [in a new window]   Figure 6. IGFBP synthesis by pVSMCs or nVSMCs. A, RT-PCR of IGFBP-1 to -6 in 3 isolates of untransformed pVSMCs and nVSMCs. B, Western blot analysis of IGFBP-2, -3, and -4 in nVSMC and pVSMC cell layers (CL) and media conditioned for 24 hours by each cell type (CM). C, Effect of CM on VSMC apoptosis. nVSMCs were placed in 0% FCS medium and treated with serum-free medium conditioned by plaque or nVSMCs for 24 hours±50 ng/mL IGF-1 or LR3. Cumulative cell death was recorded over the subsequent 24 hours. n=4; *P<0.05 at 24 hours vs plaque CM; #P<0.05 vs normal CM.

Reduced Expression of IGF-1R in Intimal and Fibrous-Cap VSMCs
Our data suggest that human atherosclerotic pVSMCs are more susceptible to apoptosis in part because of reduced IGF-1R expression. We therefore examined IGF-1R and IGF-1 expression in human coronary atherosclerotic plaques by immunocytochemistry (Figure 7). IGF-1 was found uniformly in VSMCs of both media and intima and in plaque macrophages. In contrast, IGF-1R was highly expressed in medial VSMCs, but had markedly lower expression in intimal VSMCs, particularly in shoulder regions and the fibrous cap. Where IGF-1R was expressed in the intima, it colocalized to macrophages. IGFBP3 was uniformly present through plaques and normal vessels (not shown).

View larger version (93K): [in this window] [in a new window]   Figure 7. Immunohistochemical analysis of a human coronary atherosclerotic plaque. A, Vessel has been double-stained for IGF-1R (brown) and actin (red) (x100 magnification). B, CD68 macrophage staining (brown) of the boxed shoulder region of the plaque (x200). C, IGF-1R (brown)/actin (red) staining of the boxed shoulder region of the plaque (x200). D, IGF-1 staining (brown) of the boxed shoulder region of the plaque (x200). E, IGF-1R (brown)/actin (red) staining of the fibrous cap region of the plaque (x400). F, IGF-1R (brown)/actin (red) staining of the media/intimal layers of the vessel wall (x400). G, Single labeling for IGF-1R alone (brown) demonstrating higher expression in the media (M) compared with the intima (I).

Discussion

VSMCs within advanced atherosclerotic plaques show increased apoptosis compared with cells from normal vessels.^{5 6} Whereas the complex microenvironment or their location within the plaque may increase VSMC apoptosis, we previously showed that increased susceptibility to apoptosis is an intrinsic property of pVSMCs. This property is stable over many passages in culture,²⁶ suggesting that the microenvironment or location is not responsible. In contrast, we demonstrate here that this property is caused in part by the resistance to IGF-1–mediated survival, via both reduced IGF-1R expression and increased IGFBP expression and secretion, preventing IGF-1 binding to IGF-1R.

Using radioligand binding assays, we find that pVSMCs and nVSMCs have similar high levels of ¹²⁵I–IGF-1 cell surface binding in the absence of insulin. Although insulin competes with some IGF-1 binding in nVSMCs, it produces no significant reduction in pVSMC binding. Thus, most ¹²⁵I–IGF-1 binding to both plaque and normal VSMCs can be attributed to type II IGF receptors and/or IGFBPs, which do not bind insulin and are not involved directly in IGF signaling. In contrast, the smaller component of ¹²⁵I–IGF-1 binding that is competed by insulin, and is therefore attributable to type 1 IGF receptors, was greatly decreased in pVSMCs compared with nVSMCs. This decreased binding could reflect a decrease in either the number or affinity of cell surface type I IGF receptors. It was not possible to resolve this issue by Scatchard analysis because of the interfering effect of other IGF-binding species. However, Western blotting of cell lysates with a specific antibody confirmed markedly reduced type I IGF-1R expression in pVSMCs compared with nVSMCs. We cannot rule out additional changes in the binding affinity of type I receptors in pVSMCs, although we are not aware of precedents for such a mechanism of decreased IGF binding. In any case, the reduced binding of IGF-1, caused at least in part by reduced type I receptor expression, would be predicted to lead to decreased IGF signaling in pVSMCs.

The mechanism for IGF-1R downregulation within pVSMCs is not clear, although recent evidence suggests that LDL accumulation may be responsible.³⁵ Thus, oxidized LDL, a major component of atherosclerotic plaque lipids, can downregulate expression of both IGF-1 and IGF-1R within VSMCs. Another candidate molecule for IGF-1R downregulation is the tumor suppressor gene p53. p53 directly represses the IGF-1R promoter^{36 37} and upregulates IGFBP-3.²⁷ Furthermore, p53 expression is increased in pVSMCs in vivo³⁸ and pVSMCs are very sensitive to p53-induced apoptosis.^{29 31} We have previously found that p53 transcriptional activity is similar in pVSMCs versus nVSMCs²⁹ ; however, this does not necessarily mean that repressive activity is also similar.

To ascertain whether reduced IGF-1R expression in pVSMCs resulted in reduced antiapoptotic signaling, we examined the Akt response to IGF-1. Akt kinase activity after IGF-1 stimulation was markedly reduced in pVSMCs at all time points after stimulation, suggesting that reduced IGF-1R expression may limit pVSMC survival. This is underscored by data showing a marked dose-response effect of IGF-1 on nVSMC survival, which is blunted in pVSMCs, suggesting saturation of limited IGF-1R–binding sites. These data also suggest that radiolabeled IGF-1 binding to the surface of pVSMCs is caused in part by IGF-1 binding to IGFBPs.

The IGF-binding proteins are a group of 6 proteins that modulate the actions of IGF-1.³⁹ IGFBPs control IGF-1 distribution between extracellular and intracellular compartments by modulating receptor interactions.⁴⁰ We show that pVSMCs express increased IGFBP-2 to -6 mRNA, and IGFBP-2, -3, and -4 are secreted into the CM. IGFBP-2 and -4 have been shown to be the major IGFBPs produced by VSMCs^{41 42 43 44} ; our data confirm these observations. pVSMCs also secrete higher levels of IGFBP-3 protein into CM compared with nVSMCs. IGFBP-3 is a very interesting protein because of its IGF-1–independent effects including apoptosis,^{45 46} which may be mediated via its own putative receptor. One explanation for increased IGFBP-3 secretion is p53, which activates the IGFBP-3 promoter²⁷ and works in conjunction with IGFBP-3 to promote apoptosis.⁴⁷ We show that pVSMC-CM increased apoptosis in nVSMCs from that observed with 24-hour nVSMC-CM; this might be partly explained by IGFBP-3 secretion inducing apoptosis. The effect of pVSMC-CM on nVSMCs was rescued partially by IGF-1 and more fully by LR3, an IGF-1 analogue that does not bind IGFBPs. This confirms that pVSMC-CM exerts its proapoptotic activity through IGFBPs, although it does not exclude the possibilities that other proapoptotic proteins are secreted by pVSMCs or that LR3 may have survival effects independent of IGF-1R. However, it does confirm that IGF-1 binding to IGF-1R is reduced in pVSMCs by both reduced IGF-1R expression and increased IGFBP sequestration of IGF-1. Whereas dysregulated IGFBP production is associated with many disease processes, such as Grave’s thyroid disease⁴⁸ and breast cancer,⁴⁹ such a mechanism has not previously been identified in atherosclerosis, although recent evidence suggests that oxidized LDL may directly promote IGFBP-2 and -4 expression in VSMCs.³⁵

To investigate the role of IGFBPs on survival signaling and IGFBP-independent effects of IGF-1, we examined the survival response induced by LR3 and Des 1,3. LR3 and Des 1,3 rescued pVSMCs in low serum, suggesting that high IGFBP production by pVSMCs is a major factor contributing to the lack of a survival response to IGF-1. However, although IGF-1 and LR3 produced very different levels of protection in pVSMCs, the kinetics and extent of Akt kinase activity in response to LR3 and IGF-1 were similar. This suggests that, although both LR3 and IGF-1 survival responses required PI 3-kinase, LR3 may elicit a survival response downstream of Akt or independent of Akt. Although a number of pathways are involved in growth factor–mediated survival,⁵⁰ we show that the survival effect of LR3 does not require ERK signaling. However, we have not excluded the possibility that Akt differentially phosphorylates Bad, caspase-9, or FKHRL1 in pVSMCs, resulting in reduced antiapoptotic signaling.

Although we find that pVSMCs show markedly reduced IGF-1R expression, these studies have been performed with cultured cells, including cell lines. In contrast, we confirm that reduced IGF-1R expression is also observed in the intima, particularly in the shoulder region and fibrous cap of human atherosclerotic plaques. This suggests that pVSMCs in vivo are resistant to the protective effect of even high levels of IGF-1. An increase in sensitivity to apoptosis of intimal VSMCs has profound significance for atherosclerotic plaque biology. Apoptosis of pVSMCs is associated with plaque rupture,⁵¹ and apoptosis of intimal cells is a major contributor to the procoagulant activity of plaques and the systemic procoagulant states seen in unstable coronary syndromes.⁵² VSMC apoptosis is also directly proinflammatory, causing monocyte recruitment to the vessel wall.⁵³ The recent observation that oxidized LDL induces IGF-1R downregulation and increases IGFBP synthesis³⁵ implies that progressive lipid accumulation in VSMC-derived foam cells within lesions will directly predispose to VSMC apoptosis with subsequent plaque rupture, vessel inflammation, and induction of a procoagulant state. In contrast, lipid lowering reduces lipid content of plaques, plaque inflammation, and VSMC apoptosis.⁵⁴ Our studies suggest that the modulation of IGF-1R activity and susceptibility to apoptosis in pVSMCs may be a further mechanism of action of lipid-lowering drugs.

In conclusion, we have shown that human atherosclerotic pVSMCs have reduced IGF-1R and increased IGFBP expression, both of which block the antiapoptotic action of IGF-1 on these cells. Reduced IGF-1R binding of IGF-1 results in reduced IGF-1–stimulated Akt activity in pVSMCs. This deregulated IGF-1 axis may play an important role in VSMC apoptosis in atherosclerosis and subsequent clinical complications.

Acknowledgments

This work was supported by British Heart Foundation Grants FS/97024, PG/97023, PG/99078 (M.R.B.) and CH/9400 (P.L.W.).

Footnotes

Original received October 11, 2000; revision received March 15, 2001; accepted March 16, 2001.

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