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Am J Physiol Heart Circ Physiol 292: H1051-H1057, 2007. First published October 13, 2006; doi:10.1152/ajpheart.00801.2006
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Insulin-like growth factor-I receptors in atherosclerotic plaques of symptomatic and asymptomatic patients with carotid stenosis: effect of IL-12 and IFN-{gamma}

Guanghong Jia,1,2 Gang Cheng,1,2 Krish Soundararajan,3 and Devendra K. Agrawal1,2,4

Departments of 1Biomedical Science, 2Internal Medicine, 3Surgery, and 4Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska

Submitted 26 July 2006 ; accepted in final form 12 October 2006


   ABSTRACT
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METHODS
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 DISCUSSION
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The balance between apoptosis and survival of vascular smooth muscle cells (VSMCs) in the fibrous cap appears to best correlate with plaque instability or stability and is controlled by growth factors and cytokines. We recently reported the inhibition of insulin-like growth factor (IGF)-I-induced proliferation and increase in apoptosis of VSMCs by atheroma-associated cytokines. Here we assessed the expression of IGF-I receptor (IGF-IR) in atherosclerotic plaques and in plaque VSMCs of asymptomatic and symptomatic patients with carotid stenosis and examined the effect of IGF-I, IL-12, and IFN-{gamma} on the expression of IGF-IR and IGF-binding protein (IGFBP)-3 in plaque VSMCs. We observed significantly lower density of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic nuclei and increased positive immunoreactivity to IGF-IR and mRNA transcripts of endogenous IGF-I and IGF-IR in asymptomatic than in symptomatic plaque VSMCs. Positive correlation was found between apoptosis and IGF-IR expression in asymptomatic (r2 = 0.942) and symptomatic (r2 = 0.908) plaque VSMCs. The specific binding of 125I-labeled IGF-I was 3.7-fold higher in plaque VSMCs of asymptomatic patients than in symptomatic patients. IGF-I increased both IGF-IR mRNA transcripts and expression of IGFBP-3 in VSMCs of asymptomatic plaques. IL-12 and IFN-{gamma} decreased IGF-IR mRNA transcripts and further increased the expression of IGFBP-3 in asymptomatic VSMCs but had no effect in symptomatic VSMCs. These data suggest that the decreased expression of IGF-IR mRNA and increased expression of IGFBP-3 in carotid plaques of symptomatic patients could be due to atheroma-associated cytokines and this could result in plaque instability.

apoptosis; atherosclerosis; cytokine; insulin-like growth factor binding protein; vascular smooth muscle cell

THE ROLE OF CELL LOSS and apoptotic cell death in the pathophysiology of atherosclerosis has received renewed attention. Several studies have demonstrated the presence of apoptosis in human and experimental atherosclerotic plaques (11, 16). Apoptosis regulates vascular smooth muscle cell (VSMC) numbers during the pathogenesis of both early and late stages of atherosclerotic lesions and may be involved in the disruption of the fibrous cap (5). Rupture of the fibrous cap of atherosclerotic plaques is considered to be a crucial step in the development of myocardial infarction and stroke (1). Stable plaques have a thick fibrous cap composed mainly of VSMCs and extracellular matrix enveloping a necrotic lipid core. In unstable plaques, however, part of or the entire fibrous cap is thin and contains few VSMCs and more macrophages (6). Inflammation is now thought to be a key event in the initiation and progression of atherosclerosis, and there appears to be a good correlation between inflammation and plaque instability (27). The inflammatory cells that orchestrate this response within the plaque are therefore central players in this event. There is accumulation of a large population of macrophages and polyclonal lymphocytes at sites of plaque rupture, and these cells are activated and secrete numerous cytokines including interleukin (IL)-12 and interferon (IFN)-{gamma} (18).

Insulin-like growth factor (IGF)-I can prevent apoptosis, promote matrix formation, and induce migration and proliferation of VSMCs via activation of IGF-I receptors (IGF-IR) (3). However, the ultimate cellular response to IGF-I depends on the concentration of IGF-binding proteins (IGFBPs). Six different IGFBPs have been identified so far. The protein that is predominantly bound to circulating IGF (>90% in adult serum) is IGFBP-3, which exists as a 150-kDa "ternary" complex containing an additional protein termed the acid-labile subunit (30). However, it is not known whether and how stable and unstable plaques are biochemically different and how inflammatory cytokines affect plaque stability. In this study, we assessed the expression of IGF-IR in atherosclerotic plaques and the effects of IGF-I, IL-12, and IFN-{gamma} on IGF-IR and IGFBP-3 expression in plaque VSMCs from asymptomatic and symptomatic patients with carotid stenosis.


   METHODS
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RESULTS
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Carotid endarterectomy specimens and smooth muscle cell culture. Carotid endarterectomy specimens were excised after longitudinal arteriotomy, rinsed in saline to remove surface blood, placed in the University of Wisconsin solution, and transported to the laboratory within 2–3 h. According to patients' history and clinical examination, carotid endarterectomy specimens were categorized as asymptomatic or symptomatic (Table 1). The time of collection of the specimen from the patients with symptoms was within 2 wk of development of symptoms that included hemispheric transient ischemic attacks, amaurosis fugax, or stroke. The Institutional Review Board of Creighton University approved the research protocol, and informed consent was obtained from patients before the specimens were collected.


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  		Table 1. Patient characteristics

 
The carotid plaques were transversely sectioned at 2- to 3-mm intervals. Each tissue block was embedded separately into optimum cutting temperature compound (a mixture of glycols) and frozen in liquid nitrogen. Thin sections (6 µm) were cut with a Cryocut 1800 (Reichert-Jung), and cryostat sections were mounted on electrostatically coated slides and stored at –70°C before staining.

VSMCs were isolated from the plaques by the method previously reported from our laboratory (8). Briefly, after mincing and digestion of the specimen with digestion solution (containing elastase and collagenase), the isolated cells were removed by centrifugation at 900 g for 10 min at 4°C, suspended in M199SF medium, incubated at 37°C in a humidified 5% CO2 atmosphere for 10–14 days, and passaged. The subcultured strains of the cells were used between passages 3 and 6. The confluent cells showed the characteristic hill-and-valley pattern associated with spindle-shaped VSMCs. The purity of isolated VSMCs was examined with positive immunostaining to smooth muscle {alpha}-actin (MO815; Dako, Carpinteria, CA) and caldesmon (AM332–5M; Biogenex, San Ramon, CA).

In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) reaction was performed with a commercial kit (Apotag Plus peroxidase in situ apoptosis detection kit S7101; Oncor, Gaithersburg, MD). In this method, nucleotides labeled with digoxigenin were enzymatically added to the DNA by TdT enzyme. The labeled DNA was detected with an anti-digoxigenin antibody fragment. The internal negative controls were specimens in which the TdT enzyme was substituted with 16 µl of distilled water in the preparation of working-strength TdT.

The plaques were microscopically analyzed with NIH Scion Image. The cells stained with diaminobenzidine (DAB; brown stain) and hematoxylin (blue stain) were counted in four randomly selected areas in the slide, and the percentages of nuclei positive to DAB and hematoxylin were calculated. The observer was blinded to the experimental group.

Immunohistochemistry. Sections were treated with 3% peroxide in methanol for 15 min, followed by an overnight incubation with the primary antibody at 4°C. Mouse primary monoclonal antibodies directed against IGF-IR (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and smooth muscle {alpha}-actin (1:400 dilution; MO815; Dako) were used in these experiments. After rinsing with PBS, staining in the sections was detected by the rat ABC Staining System (Santa Cruz Biotechnology). The immunopositivity in plaque sections was microscopically quantified with NIH Scion Image analysis by randomly selecting four different areas of 1-mm length under the light microscope. Then the area of positive immunostaining per millimeter squared was calculated.

Radioligand binding assays. VSMCs were isolated from human atherosclerotic plaques and subcultured as described above. Cells were incubated with 5 nM 125I-labeled IGF-I for 90 min at room temperature in the binding buffer (50 mM Tris buffer, pH 7.35, 10 mM MgCl2, and 10 µM cold insulin) in the presence and absence of 1 µM unlabeled IGF-I. Specific binding of 125I-IGF-I (5 nM) was defined as the binding displaceable by 1 µM cold IGF-I. Bound ligand to the cells was separated by filtration through GF/B filters, and unbound radioligand was removed by washing of the filter with cold washing buffer. Radioactivity was measured in a gamma counter.

Conventional and quantitative real-time RT-PCR. From 1 µg of RNA, first-strand cDNA was transcribed with a commercial kit (Qiagen). The IGF-IR mRNA transcripts were quantified by real-time RT-PCR with the ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). The oligonucleotides were purchased from Integrated DNA Technologies (IDT). For human (h)IGF-IR we used the forward primer 5'-TCATGCCTTGGTCTCCTTGTCCTT-3' and the reverse primer 5'-CCACAGTTGCTGCAAGTTCTGGTT-3'; the GenBank accession code for hIGF-IR was NM000875. The primers for the GAPDH gene were obtained from PE Applied Biosystems, with the sequence for the forward primer of 5'-AGGTCGGAGTGAACGGATTTGG-3' and for the reverse primer of 5'-TCGCTCCTGGAAGATGGTGATG-3'. The real-time RT-PCR reaction was run in the 7000 Prism Sequence Detector System by using SYBR (Applied Biosystems) as a double-stranded DNA-specific binding dye. The reactions were cycled 40 times after initial denaturation (95°C, 2 min) with the following parameters: 95°C for 15 s and annealing and extension at 60°C for 1 min. The validation experiment proved the linear dependence of the threshold cycle (CT) value of both IGF-IR and GAPDH concentration and the consistency of {Delta}CT (IGF-IR average CT – GAPDH average CT) in a given sample at different RNA concentrations. Therefore, {Delta}CT was used to reflect the relative IGF-IR expression levels. To determine the effect of different stimuli on IGF-IR mRNA transcripts compared with unstimulated cells, {Delta}{Delta}CT was calculated ({Delta}{Delta}CT = {Delta}CT stimulus – {Delta}CT unstimulated cells). IGF-IR was indexed to the GAPDH using the following formula: 1/(2Formula x 100). The value of 2Formula was calculated to demonstrate fold changes in IGF-IR gene expression in stimulated cells compared with unstimulated cells.

Conventional PCR was performed in a Perkin-Elmer GeneAMP PCR System 2400 as a hot-start PCR. After initial denaturation at 95°C for 5 min, PCR amplification was performed with denaturation steps for 45 s at 95°C, annealing for 45 s at 55°C, primer extension for 60 s at 72°C, and a final extension for 5 min at 72°C. The samples were amplified for 35 cycles, which was found to be on the linear phase. beta-Actin was used as an internal standard for RNA loading. Products were separated by gel electrophoresis (1% agarose gel) and visualized with ethidium bromide staining. Primer sequences for IGF-I were 5'-TGAAGATGCACACCATGTCCTCCT-3' (forward primer) and 5'-TGCACTCCCTCTACTTGCGTTCTT-3' (reverse primer). Primer sequences for IGF-IR were 5'-TCATGCCTTGGTCTCCTTGTCCTT-3' (forward primer) and 5'-ACGTCCAAGGGAATGGAAGGAACT-3' (reverse primer). The primers were purchased from IDT, and the GenBank accession codes for IGF-I and IGF-IR were NM000618 and NM000875, respectively.

Quantitative analysis of mRNA transcripts was performed by densitometric analysis with the UVP Bioimaging system.

Western blot analysis. Protein (10–20 µg) was separated by SDS-PAGE and transferred to Immobilon P membranes (Millipore, Bedford, MA) by electrophoresis. The membranes were incubated with blocking solution containing antibodies to IGFBP-3 (Santa Cruz Biotechnology) overnight at 4°C. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated detecting reagent specific for primary antibody, and HRP activity was detected with an enhanced chemiluminescence kit (Pierce).

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed by Student's t-test or ANOVA when appropriate to analyze differences between groups. P < 0.05 was considered significant.


   RESULTS
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Detection of apoptosis in frozen sections of plaques. We examined apoptosis by in situ TUNEL in asymptomatic and symptomatic plaques. The TUNEL-positive cells in the plaques of symptomatic lesions were present in the shoulder region of the plaque, which primarily contained VSMCs, as shown by immunopositivity to smooth muscle {alpha}-actin (Fig. 1). The number of apoptotic nuclei in the plaques from asymptomatic patients was significantly lower than in the symptomatic plaques (Fig. 1).


Figure 1
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  		Fig. 1. Top: representative photograph of serial adjacent sections taken from the shoulder region of asymptomatic (AS) and symptomatic (S) plaques showing terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) immunopositivity. Black arrows are indicative of nonapoptotic nuclei, and red arrows indicate TUNEL-positive cells, which were clearly visible in smooth muscle {alpha}-actin-positive cells. Bottom: the number of apoptotic nuclei in the plaques (as the area of the staining per mm2 in 10 high power fields) from asymptomatic lesions was significantly lower than in the symptomatic plaques (P < 0.01).

 
Immunohistochemistry to localize IGF-IR and correlation between IGF-IR and apoptosis in plaque VSMCs. We examined IGF-IR expression in symptomatic and asymptomatic carotid endarterectomy specimens by immunohistochemistry. The areas in the shoulder region of carotid plaques were captured, and the mean area of immunoreactivity was calculated. The immunopositivity to IGF-IR antibody was highly expressed in the asymptomatic plaques compared with the symptomatic plaques (Fig. 2).


Figure 2
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  		Fig. 2. Top: histological serial adjacent sections taken from the shoulder region of carotid plaques. The immunohistochemistry demonstrates that insulin-like growth factor (IGF)-I receptors (IGF-IR) are expressed differently in symptomatic and asymptomatic carotid plaques. Arrows indicate IGF-IR-positive cells, which were clearly visible in smooth muscle {alpha}-actin-positive cells. Bottom: quantification of IGF-IR immunostaining (as the area of the staining per mm2 under 10 high power fields). Image analysis indicated significantly increased immunoreactivity to IGF-IR in the shoulder region of the plaques from asymptomatic patients over that in symptomatic patients. Data are expressed as means ± SE from 12 asymptomatic and 12 symptomatic patients.

 
The correlation between increased expression of protein IGF-IR and a decreased number of apoptotic cells in asymptomatic and symptomatic plaque VSMCs was investigated with NIH Scion software. There was statistically significant positive correlation between apoptosis and IGF-IR expression in asymptomatic (r2 = 0.942) and symptomatic (r2 = 0.908) plaque VSMCs (data not shown).

Endogenous IGF-I and IGF-IR expression in symptomatic and asymptomatic plaque VSMCs. We further examined mRNA expression of endogenous IGF-I and IGF-IR in cultured VSMCs from symptomatic and asymptomatic plaques by RT-PCR. The mRNA transcripts of IGF-I and IGF-IR were significantly increased in the plaque VSMCs of asymptomatic patients than in symptomatic plaque VSMCs (Fig. 3).


Figure 3
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  		Fig. 3. Top: endogenous IGF-I and IGF-IR mRNA expression in symptomatic and asymptomatic plaque vascular smooth muscle cells (VSMCs). Lane 1, DNA ladder; lanes 2 and 3, mRNA expression of IGF-I and IGF-IR in asymptomatic plaque VSMCs; lanes 4 and 5, mRNA expression of IGF-I and IGF-IR in symptomatic plaque VSMCs. Bottom: each bar represents mean ± SE value of the relative amount of IGF-I mRNA transcripts that were measured by RT-PCR in 3 independent experiments.

 
Radioligand binding to 125I-IGF-I in plaque VSMCs. To further examine the expression of IGF-IR in atherosclerotic plaques, we performed radioligand binding assays using 125I-IGF-I in plaque VSMCs. Our results showed that the specific binding of 125I-IGF-I in the plaque VSMCs of asymptomatic patients was ~3.7-fold higher than that in the symptomatic patients (P < 0.01) (Fig. 4).


Figure 4
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  		Fig. 4. Specific binding of 125I-labeled IGF-I (5 nM) in isolated VSMCs of the plaques from asymptomatic (n = 3) and symptomatic (n = 3) patients with carotid stenosis. Cells in passage 3 were used in the radioligand binding experiments. Individual values from each patient sample are shown. Horizontal bars represent mean values.

 
mRNA expression of IGF-IR in symptomatic and asymptomatic plaque VSMCs. We studied the effect of cytokines and IGF-I on mRNA transcripts of IGF-IR in plaque VSMCs. Isolated VSMCs were cultured in serum-free medium for 24 h, followed by incubation in the presence or absence of IGF-I (100 ng/ml) with or without IL-12 (100 ng/ml) and IFN-{gamma} (100 ng/ml). The IGF-IR mRNA transcripts were significantly increased by IGF-I and markedly decreased by IL-12 or IFN-{gamma} in the plaque VSMCs of asymptomatic patients. In contrast, there was no significant effect of IGF-I, IL-12, or IFN-{gamma} on IGF-IR mRNA transcripts in the plaque VSMCs of symptomatic patients (Fig. 5).


Figure 5
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  		Fig. 5. mRNA expression of IGF-IR in symptomatic and asymptomatic plaque VSMCs. Isolated VSMCs were cultured in serum-free medium for 24 h in the presence or absence of IGF-I (100 ng/ml) with or without interleukin (IL)-12 (100 ng/ml) and interferon (IFN)-{gamma} (100 ng/ml) and lysed. Each bar represents mean ± SE value of the relative amount of IGF-IR mRNA transcripts that were measured by real-time RT-PCR in 4 independent experiments. #P < 0.01 compared with control group; §P < 0.01 compared with IGF-I+IL-12 group; *P < 0.01 compared with IGF-I+IFN-{gamma} group.

 
Expression of IGFBP-3 in symptomatic and asymptomatic plaque VSMCs. VSMCs can synthesize and secrete various forms of IGFBPs, which have been shown to have multiple functions, such as carrier function in blood, storage of IGFs, and inhibition of IGF-I actions by preventing access to IGF-IR. Therefore, we investigated the effect of cytokines and IGF-I on the expression of IGFBP-3 in plaque VSMCs of symptomatic and asymptomatic patients. There was significantly increased expression of IGFBP-3 in plaque VSMCs of symptomatic patients than in asymptomatic patients (Fig. 6). There was no effect of IGF-I, IL-12 (100 ng/ml), or IFN-{gamma} (100 ng/ml) on IGFBP-3 expression in plaque VSMCs of symptomatic patients. Interestingly, IGF-I significantly increased the expression of IGFBP-3 in plaque VSMCs of asymptomatic patients (Fig. 6). Both IL-12 and IFN-{gamma} further increased IGFBP-3 expression in plaque VSMCs of asymptomatic patients (Fig. 6).


Figure 6
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  		Fig. 6. Top: representative immunoblots for IGF binding protein (IGFBP)-3 showing the expression of IGFBP-3 in symptomatic and asymptomatic plaque VSMCs. Quiescent VSMCs were treated with IGF-I (100 ng/ml) with or without IL-12 (100 ng/ml) and IFN-{gamma} (100 ng/ml) for 24 h and then lysed. Twenty micrograms of protein were loaded in each well. Bottom: each bar represents the ratio of IGFBP-3 protein to GAPDH (mean ± SE) from 4 independent experiments. #P < 0.01 compared with control group; §P < 0.05 compared with control group.

 

   DISCUSSION
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 METHODS
 RESULTS
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Patients with or without symptoms might have similar stenosis (>70%) in the carotid artery (20), but it is unclear why in one group of patients plaques rupture and break off to produce symptoms. Some studies have also shown that rupture occurs frequently in plaques containing a soft and lipid-rich core that is covered by a thin and inflamed cap of fibrous tissue (19). The ruptured caps are usually thinner, contain less collagen, and have fewer smooth muscle cells and more macrophages compared with the intact plaques (8). In the present study, we provide further evidence that inflammatory cytokines and the density of IGF-IR in the plaques may contribute greatly toward plaque stabilization.

A balance between the apoptosis and survival of VSMCs in the fibrous cap appears to best correlate to plaque instability or stability (17). Our studies have previously shown (9) that the area of macrophage infiltration, as defined by positive immunoreactivity to CD68, in the base of the lesion and fibrous caps of symptomatic plaques was significantly greater than that in asymptomatic plaques. Because macrophages can release cytokines such as IL-12, IL-15, IL-18, and IFN-{gamma} that can induce VSMCs to undergo apoptosis, which in turn may destabilize the lesion (10), this could be a reason that the symptomatic plaque cells are more susceptible to apoptosis than asymptomatic plaques. However, apoptosis of VSMCs and macrophages that contribute greatly toward maintenance of a healthy fibrous cap is controlled by growth factors, cytokines, and mitogens to VSMCs (28). One of the factors, IGF-I, which was previously known as sulfation factor or somatomedin C, is a single-chain polypeptide of 70 amino acids that plays a role in cellular growth and survival in cardiovascular tissues through endocrine and autocrine/paracrine mechanisms (14). Acting through the IGF-IR, IGF-I prevents apoptosis, promotes matrix formation, and induces the migration and proliferation of VSMCs (15). In our previous research we demonstrated (14) that IGF-I was more potent in inducing the survival of VSMCs from the endarterectomy specimens of asymptomatic patients compared with those of symptomatic subjects, and cytokines associated with atheroma lesions decreased the activity of IGF-I-induced survival in the VSMCs of asymptomatic plaques. We further found in the present research that the area of immunoreactivity to IGF-IR in plaques and mRNA expression of endogenous IGF-I and IGF-IR in plaque VSMCs were significantly greater in asymptomatic subjects compared with symptomatic subjects. Data from other laboratories also demonstrated the role of IGF-I and IGF-IR in the pathogenesis of atherosclerotic plaques. Okura et al. (22) observed very poor expression of IGF-I and IGF-IR in the deep intima of early lesions and intima regions of advanced plaques with macrophage infiltration. In this study, apoptotic TUNEL-positive VSMCs had decreased or undetectable levels of IGF-I and IGF-IR that were not further lowered by cytokines and apoptosis-related proteins. This suggests that poor expression of IGF-I and IGF-IR in the areas with macrophage infiltration might be involved in triggering VSMC apoptosis, leading to plaque instability (22). This correlates with our radioligand binding data, which showed that the specific binding of 125I-IGF-I in the plaque VSMCs of asymptomatic patients was markedly higher than in symptomatic patients. Since we performed radioligand binding assays in the presence of 10 µM cold insulin, the specific binding of 125I-IGF-I was only to IGF-IR.

There was a dramatic upregulation of IGF-IR mRNA transcripts due to IGF-I treatment in plaque VSMCs of asymptomatic patients. However, IL-12 or IFN-{gamma} markedly suppressed IGF-IR mRNA expression in cultured VSMCs of asymptomatic plaques. The upregulation of IGF-IR expression on VSMCs by growth factors including IGF-I is critical for their survival and mitogenic effects (7). The downregulation of IGF-IR expression by oxidized LDL and cytokines is critical for their apoptosis, but the mechanism is not clear (25). Recent evidence suggests that the tumor suppressor gene p53 may be responsible by directly repressing the IGF-IR promoter and upregulating IGFBP-3 (23). Furthermore, p53 expression was increased in VSMCs, making them sensitive to p53-induced apoptosis (23).

The biological activity of IGF-I is regulated partly by its association with IGFBPs, which act as reservoirs for IGF-I and modulate IGF-I interactions with the cell membrane receptor (26). Although evidence suggests that individual species of binding proteins may both inhibit and facilitate IGF-I action, the exact role of each IGFBP remains largely unknown (24). Studies showed that IGFBP-3, which is the most abundant carrier protein for IGF-I, may also function as a negative regulator of cell growth and act as a proapoptotic factor (2). In fibroblasts, transfection of IGFBP-3 into IGF-IR-negative fibroblasts inhibited the proliferative response to serum (29), and exogenous IGFBP-3 inhibited growth of human breast cancer cells (21). In VSMCs TNF-{alpha} reduced IGF-I, increased IGFBP-3 expression, and led to a reduction in bioactive IGF-I by a direct effect (2). Our findings have far-reaching implications for understanding the mechanisms of IGF-I and cytokines in apoptosis and proliferation of asymptomatic and symptomatic plaque VSMCs. These results showed that IL-12 and IFN-{gamma} markedly increased the expression of IGFBP-3 in plaque VSMCs of asymptomatic patients. However, IGF-I also significantly increased the expression of IGFBP-3, which is negative to the proliferation and survival of VSMCs in asymptomatic plaques. This could be an endogenous regulatory mechanism to regulate the effect of IGF-I. Compared with IGF-I actions, the effects of IGFBP-3 are weaker on the survival and apoptosis of atherosclerotic VSMCs. Nevertheless, our data correlate with those of Hayford and colleagues (13), who also reported that IGF-I increased IGFBP-3 expression and induced proliferation in human VSMCs at the same time. IGFBP-3 is an interesting protein because of its IGF-I-independent effects including apoptosis, which may be mediated via its own putative receptor (23). One explanation for increased IGFBP-3 secretion is p53, which activates the IGFBP-3 promoter (4) and works in conjunction with IGFBP-3 to promote apoptosis (12).

Delafontaine and colleagues (7) demonstrated the presence of IGFBP-2 through -6 in human VSMCs, and these IGFBPs were markedly increased in atherectomy specimens. However, IGF-I and IGF-IR expression was reduced in advanced plaques, and this was consistent with the increased apoptotic rate of VSMCs from atherosclerotic plaques, which also secrete high levels of IGFBPs. This suggests the potential role of all IGFBPs, at least in part, at the level of local atherosclerotic lesion. Since IGFBP-3 is the most abundant carrier protein for IGF-I in VSMCs, we focused on the expression and regulation of only IGFBP-3. However, the potential role of other IGFBPs in IGF-I and IGF-IR function in symptomatic and asymptomatic plaques warrants careful analysis.

As indicated by our findings in the plaque VSMCs of symptomatic patients, IGF-I in the absence or presence of IL-12 or IFN-{gamma} did not significantly affect either mRNA transcripts of IGF-IR or IGFBP-3 protein expression. This could be explained if the VSMCs in the symptomatic plaques were already exposed to endogenously released IL-12 and IFN-{gamma} from infiltrated macrophages and lymphocytes, respectively, in atherosclerotic plaques. This might have attenuated the endogenous expression of IGF-IR mRNA transcripts and IGFBP-3 to such an extent that further addition of exogenous cytokines had no effect. Indeed, we and other investigators (9, 20) have reported a significantly increased number of inflammatory cells in symptomatic plaques over that in asymptomatic plaques.

In summary, data from this study demonstrate greater expression of IGF-IR in atherosclerotic plaques of asymptomatic patients than in symptomatic patients and demonstrate that cytokines associated with atheromatous plaques decrease the density of IGF-IR and increase IGFBP-3 in the VSMCs of asymptomatic plaques. This could be a potential mechanism underlying instability of atheromatous plaques in symptomatic patients.


   GRANTS
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This study was supported by National Heart, Lung, and Blood Institute Grants R01-HL-070885 (D. K. Agrawal) and R01-HL-073349 (D. K. Agrawal). Its contents are solely the responsibility of the authors, and no conflicts of interest exist for any of the authors.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Agrawal, Creighton Univ. School of Medicine, CRISS II Rm. 510, 2500 California Plaza, Omaha, NE 68178 (e-mail: dkagr{at}creighton.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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