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Cardiovascular Research 2004 61(2):247-255; doi:10.1016/j.cardiores.2003.11.008
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Copyright © 2004, European Society of Cardiology

Native and oxidized low density lipoproteins oppositely modulate the effects of insulin-like growth factor I on VSMC

B González-Timóna, M González-Muñozb, C Zaragozac,d, S Lamasa,* and E.M Melián

aEndocrinology Division, Hospital Carlos III, Instituto de Salud Carlos III, Comunidad de Madrid, C/ Sinesio Delgado 10, Madrid 28029, Spain
bImmunology Division, Hospital Carlos III, Instituto de Salud Carlos III, Madrid 28029, Spain
cCentro de Investigaciones Biológicas, Instituto "Reina Sofía" de Investigaciones Nefrológicas, Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain
dCentro Nacional de Investigaciónes Cardiovasculares, CNIC, Madrid 28029, Spain

* Corresponding author. Tel.: +34-91-453-2500x2736; fax: +34-91-733-6614. emelian.hciii{at}salud.madrid.org

Received 25 September 2003; revised 27 October 2003; accepted 14 November 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Changes in the local expression and signaling activity of the insulin-like growth factor-I (IGF-I) axis regulate growth and survival of plaque-derived vascular smooth muscle cells (VSMC) and influence plaque fate. Recent evidence suggests that accumulation of low density lipoproteins (LDL) in VSMC during the progression of atherogenesis is linked to local changes in IGF-I signaling. We investigated the effects of LDL on the biological actions and downstream signaling pathways mediated by this growth factor in A10 VSMC. Methods and Results: We first characterized the effects of LDL on the proliferative and anti-apoptotic actions of IGF-I in A10 VSMC. Native LDL were mitogenic and synergistically enhanced DNA synthesis induced by IGF-I from 4-, 9- up to 7.8-fold, while having no effect on its anti-apoptotic actions. In contrast, oxidized LDL, at oxidation levels that did not modify these actions by themselves, significantly reduced the mitogenic and survival effects of IGF-I by 40% and 60%, respectively. These observations correlated with opposite changes exerted by native and oxidized LDL on the insulin receptor substrate-1 (IRS)-associated PI3 kinase/Akt response to IGF-I. The extracellular signal-regulated kinase (ERK) signaling response was not affected. Conclusions: Our study demonstrates a previously unidentified modulation of the actions of IGF-I on A10 VSMC by LDL, dependent on their extent of oxidative modification. Our findings suggest that the differential modulation of the PI3 kinase/Akt response to IGF-I play a pivotal role.

KEYWORDS Atherosclerosis; Growth factor; Lipoprotein; Signal transduction; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Phenotypic modulation of vascular smooth muscle cells (VSMC) is critical to the progression of atherosclerosis [1]. It has recently been shown in humans that local expression of insulin-like growth factor (IGF-I) and IGF-I receptor (IGF-IR) varies with the phenotypic state of plaque-derived VSMC [2]. Thus, cells from early lesions show a significant up-regulation of this axis, in contrast to the clear reduction of IGF-I and IGF-IR expression in VSMC from advanced atherosclerotic lesions [3]. Moreover, the impairment of the IGF-I signaling system in human VSMC from advanced plaques is associated with an increase in their intrinsic sensitivity to apoptosis, with subsequent cell loss and plaque instability [4].

High serum low density lipoproteins (LDL) levels are a well-established risk factor for atherosclerosis [5], and local oxidation of LDL in the atheroma plaque plays a significant pathogenic role in lesion stability, in part through changing VSMC phenotype and function [1,6,7]. In VSMC from human atherosclerotic plaques, there is a strong correlation between macrophage infiltration, weak IGF-I and IGF-IR expression, and increased apoptosis [3]. In the rats, mitogenic native LDL (nLDL) increases the expression of IGF-I and IGF-IR, whereas pro-apoptotic oxidized LDL (oxLDL) has the opposite effect [8]. Based on these findings, it has been suggested that LDL accumulation and modification in plaque-derived VSMC may regulate local IGF-I synthesis. However, the possible impact of these lipoproteins on IGF-I intracellular pathways downstream to this axis expression remains unexplored.

A10 VSMC are a model of neointimal VSMC [9] that is useful for evaluating interactions between LDL and IGF-I axis expression [10]. We have previously shown that activation of PI3 kinase by IGF-I is required for IGF-I-induced mitogenesis of A10 VSMC and, furthermore, that cooperation between nLDL and IGF-I in A10 proliferation is associated with the up-regulation by nLDL of IGF-I-induced insulin receptor substrate-1 (IRS-1) dependent PI3 kinase/Akt activation [11]. In the present study, we show that nLDL and oxLDL differentially affect the mitogenic and anti-apoptotic actions of IGF-I in A10 VSMC. We furthermore demonstrate a good correlation between the aforementioned effects and the underlying opposite modulation by nLDL and oxLDL of IGF-I-induced PI3 kinase/Akt activity associated with IRS-1.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Materials
All chemicals and reagents were purchased from Sigma (Madrid, Spain) unless specified otherwise. Human IGF-I was purchased from R&D systems (Minneapolis, MN). PY20 antiphosphotyrosine was from Santa Cruz Biotechnology, (California). IRS-1 antibody was from Upstate (New York, NY). Phosphospecific activated and control antibodies for PKB/Akt, ERK 1/2 kinases, and HRP-conjugated anti-rabbit were from New England Biolabs (Beverly, MA). HRP-linked anti-mouse antibodies were from Dako (Glostrup, Denmark). Protein A-Sepharose 6MB and isotopes were from Amersham Pharmacia Biotech (Barcelona, Spain). LY294002 and PD98059 were from Calbiochem (Darmstadt, Germany). Fetal bovine serum (FBS), DMEM, and antibiotics were from BioWhittaker (Walkersville, MD).

2.2. Lipoprotein preparation and oxidative modification
Lipoproteins were isolated by sequential ultracentrifugation (LDL d = 1.019–1.063 g/ml) of EDTA-anticoagulated plasma obtained from healthy normolipemic volunteers [12]. The presence of potential contaminant IGF-I was excluded by Western blot. For oxidation procedures, LDL were dialyzed against EDTA-free phosphate-buffered saline for 24 h to remove the EDTA, and then incubated with 5 µM CuSO4 for 24 h at 37 °C. Oxidation was terminated by adding 10 mM EDTA and filtering the sample. Lipid peroxidation products of oxLDL preparations were determined by measuring thiobarbituric acid reactive substance (TBARS), and values were expressed as nanomolar equivalents of malondialdehyde (MDA) per milligram LDL protein [13]. Protein concentrations of lipoprotein preparations were determined by the Lowry method. The mean TBARS (nmol/mg LDL protein) for six separate preparations was ≤1 for native LDL (nLDL), 10–15 for mildly oxidized (moxLDL), 20–25 for oxidized (oxLDL), and 30–35 for highly oxidized LDL (hoxLDL). All lipoproteins were stored at 4 °C and used within 2 weeks after preparation.

2.3. Cell culture
Rat A10 VSMC derived from thoracic aorta of fetal rats were prepared as previously described [11]. Passages 20 through 30 were used for experiments. In order to synchronize cells (growth arrest) for [3H]-thymidine and short-term stimulation experiments, the medium was changed to serum-free DMEM (SFM) 48 h before stimulus. This SFM was replaced with SFM together with the appropriate stimulus for various times as indicated.

2.4. [3H]-thymidine incorporation assay
DNA synthesis was measured as previously described [11].

2.5. Apoptosis measurement
Apoptosis was determined by flow cytometry using the "Annexin V-FITC Apoptosis Detection Kit I" (BD Biosciences, San Diego, CA), according to the manufacturer's instructions. Briefly, VSMC were grown in six-well plates and incubated with IGF-I and/or lipoproteins in SFM for 24 h. After removing the supernatant, cells attached to the plate were washed once with PBS, collected with 0.25% trypsin EDTA, and combined with detached cells present in the supernatant. VSMC were spun, resuspended with binding buffer, and incubated with FITC-Annexin V and PI for 20 min at room temperature. Flow cytometry analysis was performed immediately after staining.

2.6. Western blot analysis
After incubations with IGF-I and/or LDL, cells were washed with cold PBS, lysed, and subjected to SDS-PAGE as reported previously [11]. Alternatively, IRS-1 was immunoprecipitated from 1.5 mg of total cell protein and analyzed by Western blot with anti-phosphotyrosine [11]. Protein concentration was determined by the Bradford method.

2.7. Assay of PI3 kinase activity
PI3 kinase activity was assayed as described [14]. In brief, cells were washed, lysed, and incubated with IRS-1 antibody followed by incubation with protein-A Sepharose. After washing, the activity of PI3 kinase present in the resuspended immunoprecipitate was determined with phosphatidylinositol (20 µg) and [{gamma}-32P] ATP.

2.8. Statistical analysis
Representative experiments of three to four independently conducted or the mean±SEM for all data are shown. Unpaired Student's t-test or appropriate non-parametric tests were used for analysis of differences between various treatments. P values ≤0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Native and oxidized LDL differentially modulate IGF-I-induced regulation of A10 VSMC growth
The oxidative state of LDL has been shown to be important for their apoptotic and/or proliferative actions in the vascular wall [15], so we first examined the effects of modifying LDL oxidation on the previously characterized IGF-I-induced synthesis of DNA in A10 VSMC (Fig. 1A). Sixteen hours exposure to 5 nM IGF-I induced a 4.9±0.2-fold increase in [3H]-thymidine incorporation vs. control cells (P<0.001). Native LDL increased basal [3H]-thymidine incorporation by themselves, whereas moxLDL and oxLDL alone had no significant effects on DNA synthesis. However, the presence of nLDL and moxLDL enhanced the response to IGF-I in a synergistic manner (from 4.9-fold with IGF-I alone up to 7.8±0.7-fold with nLDL, P<0.001; and up to 6.9±0.45-fold with moxLDL, P<0.01). By contrast, oxLDL exerted a marked inhibitory effect (from 4.9-fold with IGF-I alone down to 3±0.1-fold with oxLDL, P<0.001). This inhibition was more pronounced the higher the degree of LDL oxidation, as measured by the TBARS content, but this phenomenon was also concentration-dependent. While no oxLDL dose changed basal DNA synthesis (data not shown), lower concentrations of oxLDL (5 and 15 µg/ml) enhanced IGF-I-induced mitogenesis, whereas higher concentrations (50 µg/ml) were inhibitory (Fig. 1B). Long exposure (16 h) of A10 cells in serum-free medium to hoxLDL was cell-toxic, with a corresponding decrease in basal [3H]-thymidine incorporation, so this treatment was excluded from subsequent long incubation experiments.


Figure 1
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  		Fig. 1 Oxidation level and concentration determine the effect of LDL on IGF-I-induced A10 DNA synthesis. Effects of LDL on basal and IGF-I induced [3H]-thymidine incorporation in A10 VSMC as a function of the LDL oxidative state (TBARS content) (A) or the concentration of oxLDL (B). After synchronization, cells were exposed to 5 nM IGF-I or vehicle in the presence or absence of LDL. [3H]-thymidine was present for the last 2 h, and its incorporation (cpm/well) was measured 16 h after addition of IGF-I or vehicle. In both A and B panels, values represent mean±SEM (n = 5). Where error bars are not visible, they are smaller than data points. (*) P<0.01 and (**) P<0.001 vs. control. ({dagger}) P<0.05; ({dagger}{dagger}) P<0.01 and ({dagger}{dagger}{dagger}) P<0.001 vs. IGF-I alone. (a) P<0.01 vs. the theoretical additive effect of IGF-I plus each lipoprotein, suggesting a synergistic action of these factors on DNA synthesis when added together. nLDL, native LDL, moxLDL, mildly oxidized LDL, oxLDL oxidized LDL, and hoxLDL, highly oxidized LDL.

 
We next analyzed the effect and pathways involved on IGF-I-induced survival of A10 VSMC, and the possible modulation by LDL. As seen in Fig. 2A, the presence of IGF-I markedly reduced the proportion of cells in apoptosis after 24 h of treatment (IGF-I treated cells 22.2±1, 5% vs. 100% control values, P<0.001). Both LY294002 (a specific and irreversible PI3 kinase inhibitor) and PD98059 (a specific inhibitor of the ERK 1/2 pathway), significantly inhibited the IGF-I promotion of cell survival (LY2940002, 20 µM: 50.1±9 apoptosis, PD98059, 20 µM: 74.8±6.5% apoptosis; P<0.01 and P<0.001 vs. IGF-I value, respectively), without affecting basal apoptosis levels. Simultaneous blockade of both pathways induced apoptosis in control cells, and this effect was additive in IGF-I treated cells (99.5±6% apoptosis, P<0.001 vs. IGF-I value). Incubation with nLDL or oxLDL alone for 24 h did not induce apoptosis beyond control levels, but oxLDL presence significantly inhibited the IGF-I protective effect (71±4% vs. 29±7% with IGF-I alone, P<0.001) (Fig. 2B). The data presented in Figs. 1 and 2BGo indicate that both the degree of oxidation and the LDL concentration are important factors determining the nature of their interaction with the IGF-I axis in A10 VSMC.


Figure 2
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  		Fig. 2 OxLDL and inhibitors of PI3 kinase and ERK 1/2 reduce the protective effect of IGF-I against apoptosis in A10 VSMC. (A) Influence of PI3 kinase and ERK 1/2 on the IGF-I-induced protection against apoptosis. Serum-free, sub-confluent (50–60%) A10 VSMC were treated with vehicle (white bars) or 5 nM IGF-I (gray bars): either alone (basal), or after pretreatment for 30 min with 20 µM LY294002 (inhibitor of PI3 kinase) or 20 µM PD98059 (inhibitor of ERK 1/2), or a combination of these two agents. Apoptosis was assessed after 24 h by flow-cytometric analysis of cells that were simultaneously stained with Annexin V and propidium iodide (PI). The dotplots show a representative experiment, and the bar chart shows the statistical analysis of the results of four experiments. In the dotplots, the proportion on cells in each quadrant is given as the percentage of forward- and side-scatter gated cells. Annexin V-positive/PI-negative cells (lower right quadrant) are in early apoptosis. In the bar chart, results are expressed as the percentage of the reading for the basal control condition (C). Each column represents the mean±SEM (n = 4). (*) P<0.001 vs. control; ({dagger}) P<0.01 and ({dagger}{dagger}) P<0.001 vs. IGF-I alone. (B) Effects of LDL on the protective effect of IGF-I against apoptosis. Serum-free sub-confluent (50–60%) A10 VSMC were treated with vehicle (white bars) or 5 nM IGF-I (gray bars), in the presence or absence of LDL for 24 h. Apoptosis was determined relative to the basal control condition (C) as above. Each column represents the mean±SEM (n = 5). (*) P<0.001 vs. control; ({dagger}) P<0.001 vs. IGF-I alone.

 
3.2. Native and oxidized LDL differentially interact with IGF-I-stimulated pathways in A10 VSMC
Activation of the PI3 kinase/Akt pathway by IGF-I is required for most of its proliferative effects on A10 VSMC [11], and for part of its anti-apoptotic effects (see Fig. 2). We hypothesized that nLDL and oxLDL differentially regulate the biological response to IGF-I by acting on the PI3 kinase/Akt signaling pathway. Neither LDL type was able to induce Akt phosphorylation alone. However, when added together with IGF-I, nLDL and oxLDL had opposite effects on the Akt response. Whereas nLDL up-regulated IGF-I-mediated Akt phosphorylation (123±4 vs. 100±2 with IGF-I alone, P<0.01), oxLDL was inhibitory (77±11 vs. 100±2 with IGF-I alone, P<0.01) (Fig. 3A). Differences were highly significant between the IGF-I+nLDL and the IGF-I+oxLDL experimental groups (P<0.001). The effect of oxLDL on IGF-I-mediated Akt activation was concentration-dependent with only the 50 and 100 µg/ml doses being inhibitory (Fig. 3B), resembling the modulation by oxLDL of IGF-I-dependent [3H]-thymidine incorporation (see above).


Figure 3
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  		Fig. 3 Differential effects of nLDL and oxLDL on IGF-I-induced phosphorylation of Akt. (A) Representative blot and statistical analysis of the effects of LDL on basal and IGF-I-induced Akt phosphorylation. After synchronization, cells were treated for 10 min with 5 nM IGF-I in the presence or absence of nLDL or oxLDL (50 µg/ml). Akt activation was revealed by Western blot for phosphorylated Akt (p-Akt). The chart shows the ratios of the band intensities for phosphorylated and total Akt (total) expressed as a percentage of the values obtained for treatment with IGF-I alone. Each column represents the mean±SEM (n = 8). ({dagger}) P<0.01 vs. IGF-I alone; (#) P<0.001 IGF-I+nLDL vs. IGF-I+oxLDL. (B) Concentration-dependence of oxLDL effect on IGF-I induced Akt phosphorylation. Representative blot of the effects of different concentrations of oxLDL on IGF-I-induced Akt phosphorylation. After synchronization, cells were treated for 10 min with 5 nM IGF-I in the presence or absence of different concentrations of oxLDL. Akt activation was revealed by Western blot for phosphorylated Akt (p-Akt) and total Akt (Total). A representative blot from two is shown. C=control cells.

 
We also studied the ERK 1/2 pathway, which is known to be activated by LDL in VSMC [16]. In our system, both LDL forms were able to increase ERK 1 and ERK 2 phosphorylation, but the effects of IGF-I on ERK phosphorylation were not significantly different in the presence of LDL (Fig. 4). Time-course experiments with oxLDL showed a similar patter to nLDL [11]: that is a small additive effect on ERK phosphorylation at times when their isolated effects on this pathway decline (data not shown).


Figure 4
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  		Fig. 4 Native and oxidized LDL do not modify IGF-I-induced ERK phosphorylation in A10 VSMC. Representative blot and statistical analysis of the effects of LDL on basal and IGF-I-induced ERK 1/2 phosphorylation. After synchronization, cells were treated for 10 min with 5 nM IGF-I in the presence or absence of nLDL or oxLDL (50 µg/ml), and ERK activation was measured by Western blot for phosphorylated ERK 1 and 2 (P-ERK 1/2). The chart shows the ratios of the band intensities for phosphorylated and total ERK (total) expressed as a percentage of the values obtained for treatment with IGF-I alone. Each column represents the mean±SEM (n = 4). (*) P<0.01 vs. control (white bar).

 
Taken together, these findings suggest a direct and pivotal role for the PI3 kinase/Akt pathway, but not the ERK pathway, regulating the cross-talk between LDL and IGF-I signaling in A10 VSMC.

3.3. Native LDL up-regulates and oxidized LDL down-regulate IGF-I-induced PI3 kinase activation associated with IRS-I in A10 VSMC
We previously showed that nLDL up-regulate PI3 kinase activity associated to IRS-1 in cells treated with IGF-I, while not modifying this activity by themselves [11]. We therefore tested whether oxLDL could affect the Akt response to IGF-I by inhibiting this early step in the PI3 kinase/Akt pathway. Whereas nLDL significantly increased IRS-1-associated PI3 kinase activity in response to IGF-I, oxLDL significantly decreased it (nLDL: 163±11% vs. 100% for IGF-I alone, P<0.001; oxLDL: 73±3% vs. 100% for IGF-I alone, P<0.01) (Fig. 5B). Once again, differences between experimental groups were highly significant when comparing IGF-I+nLDL vs. the IGF-I+oxLDL (P<0.001). Even in these short exposure experiments, hoxLDL presence was associated with a more prominent inhibition of IGF-I-induced PI3K kinase activity than oxLDL (hoxLDL: 38±13% vs. 100% for IGF-I alone, P<0.001).


Figure 5
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  		Fig. 5 The LDL oxidation level differentially modulates IRS-1-associated PI3 kinase activity induced by IGF-I in A10 VSMC. (A) Representative autoradiogram and statistical analysis of the effects of LDL on IRS-1-associated PI3 kinase activity basal and induced by IGF-I. After synchronization, cells were treated for 3 min with vehicle or 50 µg/ml of LDL (nLDL or oxLDL) before stimulation with 5 nM IGF-I. Cells were lysed 5 min after IGF-I addition, and IRS-1 was immunoprecipitated and assayed for PI3 kinase activity. (*PIP) [32P] phosphatidylinositol phosphate. Results are expressed as a percentage of the band intensity obtained with IGF-I alone. Each column represents the mean±SEM (n = 6). ({dagger}) P<0.001 vs. IGF-I alone; (#) P<0.001 IGF-I+nLDL vs. IGF-I+oxLDL or IGF-I+hoxLDL. (B) Representative blot and statistical analysis of the effects of LDL on IGF-I-stimulated tyrosine phosphorylation of IRS-1. After synchronization, cells were incubated with or without 50 µg/ml of oxLDL for 3 min before the addition of IGF-I (5 nM) or vehicle. Cells were lysed 5 min after IGF-I addition, immunoprecipitated with IRS-1 antibody, separated by 10% SDS-PAGE, transferred to PVDF, and blotted with an antibody against phosphotyrosine ({alpha}PY). Results are expressed as a percentage of the band intensity obtained with IGF-I alone. PY, Phosphotyrosine; IP, immunoprecipitating; IB: immunoblotting. Values are the mean±SEM (n = 3). (*) P<0.001 vs. control; ({dagger}) P<0.001 vs. IGF-I and (#) P<0.001 IGF-I+nLDL vs. IGF-I+oxLDL.

 
Changes to the tyrosine phosphorylation of IRS proteins could modulate their docking to the p85 subunit of PI3 kinase, and could explain the divergent effects of LDL on IRS-I associated PI3 kinase activity induced by IGF-I [17]. As expected, IGF-I increased the tyrosine phosphorylation of IRS-I by 5-fold (Fig. 5B). Pretreatment of A10 VSMC for 3 min with 50 µg/ml nLDL did not significantly alter the IGF-I effect but, when oxLDL was present, IGF-I failed to induce IRS-1 tyrosine phosphorylation (37±11% vs. 100% for IGF-I), pointing towards an early interference of oxLDL in the IGF-I signaling pathway.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The balance between proliferation and apoptosis of VSMC plays a critical role in the progression of the atheroma plaque [18]. During this process, arterial VSMC migrate from the media to the intima of the vascular wall, shifting from a contractile to a proliferative phenotype and modifying their response to environmental stimuli [1,19]. In general, this proceeds so that a high percentage of VSMC die from apoptosis, leading to potential deleterious consequences resulting from weakened and destabilized atheroma plaques [18].

In contrast with nLDL, which are known to be mitogenic and non-apoptotic in human vascular cells, oxLDL induce apoptosis by promoting the expression of pro-apoptotic proteins in VSMC from advanced plaques [3]. Moreover, high local and plasma levels of oxLDL are correlated in humans with vulnerability to rupture of atherosclerotic lesions [6]. In vitro, native and oxidized LDL have opposite effects on VSMC growth and survival [15], but the underlying mechanisms remain poorly understood.

An important finding in our study is that nLDL and oxLDL induce different responses to IGF-I in A10 VSMC. These effects are associated with specific interactions with the IGF-I signaling system, and are dependent on the oxidative state and concentration of LDL. Significantly, the effects of oxLDL on the IGF-I axis precede the observation of direct deleterious actions of these lipoproteins on mitogenesis and apoptosis of A10 VSMC. Given the essential role of IGF-I in cell cycle progression and survival of VSMC [20], the coexistence of a local regulation of IGF-I/IGF-IR synthesis [3,21–23] with a specific regulation of the IGF-I signaling system by lipoproteins could contribute to VSMC proliferation and/or apoptosis and hence affect atherosclerotic plaque stability.

IGF-I exerts its effects by interacting with IGF-IR, whose expression varies with the cellular growth status [24]. IGF-IR is a transmembrane tyrosine kinase that activates two primary intracellular pathways: PI3 kinase and extracellular signal-regulated MAPK (ERK1/2) [20]. Previous reports from our laboratory and others indicate that PI3 kinase/Akt activity associated to IRS-1 is preferentially involved in the proliferative actions of IGF-I in VSMC [11,25–27]. Downstream targets that mediated the anti-apoptotic actions of IGF-I in VSMC are less clear, although reported observations suggest the involvement of multiple signal transduction pathways [26,28]. Our current study shows that IGF-I has a relevant protective effect on A10 cell survival. However, it appears in this case that activation of the PI3 kinase/Akt pathway is only partially responsible for this effect. This is in keeping with studies on VSMC from human plaques, where the suppression of the PI3 kinase/Akt or ERK pathways only incompletely prevents the anti-apoptotic effects of IGF-I [4,26].

To our knowledge, our data provide the first proof of the modulation by LDL of IGF-I-induced responses and intracellular pathways in VSMC. The PI3 kinase signaling pathway seems to play a critical role in the pathological accumulation of VSMC observed in various types of vascular lesions [29]. More specifically, IGF-I-associated PI3 kinase activation has been reported to be central to IGF-I's effects on VSMC proliferation and migration [27], VSMC apoptosis prevention [30,31] and VSMC phenotype differentiation [32]. The intrinsic in vivo sensitivity of human VSMC to apoptosis is associated with an impairment in the increase in Akt kinase activity provoked by IGF-I [4]. A dual mechanism, in which LDL, as function of their oxidative modification in the vascular wall, initially favor and later impair IGF-I-mediated PI3 kinase/Akt activation could therefore play a critical role in VSMC accumulation and depletion during progression of atherogenesis [18,33]. Several facts lead us to believe this to be the case in A10 VSMC. First, LDL alone does not induce IRS-I-associated PI3 kinase or Akt phosphorylation. Hence, a specific regulation of the PI3 kinase/IRS-1 interaction after IGF-I stimulation is a plausible scenario. Second, the potentiation and inhibition by LDL of the IRS-I-associated PI3 kinase/Akt response to IGF-I closely parallel the respective modulations of IGF-I induced DNA synthesis. Third, there is a tight match in the extents to which oxLDL and LY294002 restrain the survival effects of IGF-I (2.3 times vs. IGF-I values). Last, ERK 1/2 activation in response to IGF-I does not vary between nLDL and oxLDL treatments even though both LDL activate this pathway.

A large body of published research has shown that, beyond local lipid accumulation, the mechanism by which nLDL and oxLDL regulate the vascular behavior of cell types involved in atherosclerosis largely depends on the degree of oxidation, the exposure time, and the concentration [15,18,33–35]. In A10 VSMC cells, nLDL induce IGF-IR expression [10], and additional data from rat VSMC also support the strength of the relationship between non-modified LDL and IGF-I signaling pathways; thus, nLDL-induced DNA synthesis is prevented with anti-IGF-I antiserum [8]. In addition, studies with statins have demonstrated the specific requirement for metabolic intermediates of the cholesterol biosynthetic pathway in the activation of cellular proliferation in response to IGF-I [36].

In cells treated with oxLDL, the inhibition of the IGF-I response at an early stage suggests that a decrease in the intrinsic tyrosine kinase activity of IGF-IR could inhibit p85 binding [37]. Preliminary data implicate PKC in the oxLDL effects in our system, as PKC inhibitors rescue the inhibition caused by oxLDL on Akt phosphorylation after IGF-I stimulus (data not shown). The PKC pathway plays an essential role in A10 growth [38], and oxLDL has been shown to activate PKC in other models of VSMC [16]. Moreover, PKC inhibitors prevent the inhibition on IGF-IR/IRS tyrosine phosphorylation induced by other specific down-regulators of insulin-dependent PI3 kinase activation [14,39,40].

The results herein reported regarding the interaction between IGF-I and LDL could represent a model mechanism in the cooperation of LDL with growth-promoting factors for VSMC. LDL enhance the expression of several classic growth factors or their receptors, as PDGF [41], angiotensin II [42] or thrombin [43], and it is conceivable that these lipoproteins will modify specific signaling routes depending on the growth factor targeted.

In summary, changes in the concentration and oxidative state of LDL lead to different biological outcomes for IGF-I-induced mitogenesis and survival of A10 VSMC, and these effects are associated with an opposite regulation by nLDL and oxLDL of the IGF-I-induced PI3 kinase/Akt activity associated to IRS-I. These data suggest that LDL could be affecting the development of atherogenesis development in vivo not only by modulating IGF-I/IGF-IR expression as previously suggested, but also by altering the balance signaling pathways elicited by IGF-I in VSMC.


   Acknowledgements
 
We are grateful to Dr. A. Alvarez Barrientos for his help with flow cytometric analysis. We thank Dr. F. Sanchez-Franco for his support during the development of this work. Belén González is a predoctoral fellow from the Instituto de Salud Carlos III (99/4213). This work was supported by Grants SAF 98-0003 from the Plan Nacional de Investigación y Desarrollo and 08.4/0031.2/2000 from the Comunidad de Madrid, Spain (to EM), and by Grant SAF 2000-0149 (to SL).


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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L. B. Allen, B. E. Capps, E. C. Miller, D. R. Clemmons, and L. A. Maile
Glucose-Oxidized Low-Density Lipoproteins Enhance Insulin-Like Growth Factor I-Stimulated Smooth Muscle Cell Proliferation by Inhibiting Integrin-Associated Protein Cleavage
Endocrinology, March 1, 2009; 150(3): 1321 - 1329.
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