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Endocrinology Vol. 148, No. 5 2435-2443
Copyright © 2007 by The Endocrine Society

Hyperglycemia Alters the Responsiveness of Smooth Muscle Cells to Insulin-Like Growth Factor-I

Laura A. Maile, Byron E. Capps, Yan Ling, Gang Xi and David R. Clemmons

Division of Endocrinology, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7170

Address all correspondence and requests for reprints to: Laura A. Maile, Ph.D., 5029 Burnett Womack, CB 7170, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: laura_maile{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I stimulation of smooth muscle cell (SMC) migration and proliferation requires {alpha}Vß3 ligand occupancy. We hypothesized that changes in the levels of extracellular matrix proteins induced by alterations in glucose concentrations may regulate the ability of SMCs to respond to IGF-I. IGF-I stimulated migration and proliferation of SMCs that had been maintained in 25 mM glucose containing media, but it had no stimulatory effect when tested using SMCs that had been grown in 5 mM glucose. IGF-I stimulated an increase in Shc phosphorylation and enhanced activation of the MAPK pathway in SMCs grown in 25 mM glucose, whereas in cells maintained in 5 mM glucose, IGF-I had no effect on Shc phosphorylation, and the MAPK response to IGF-I was markedly reduced. In cells grown in 25 mM glucose, the levels of {alpha}Vß3 ligands, e.g. osteopontin, vitronectin, and thrombospondin, were all significantly increased, compared with cells grown in 5 mM glucose. The addition of these {alpha}Vß3 ligands to SMCs grown in 5 mM glucose was sufficient to permit IGF-I-stimulated Shc phosphorylation and downstream signaling. Because we have shown previously that {alpha}Vß3 ligand occupancy is required for IGF-I-stimulated Shc phosphorylation and stimulation of SMC growth, our data are consistent with a model in which 25 mM glucose stimulates increases in the concentrations of these extracellular matrix proteins, thus enhancing {alpha}Vß3 ligand occupancy, which leads to increased Shc phosphorylation and enhanced cell migration and proliferation in response to IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPOSURE OF CELLS in culture to increased levels of glucose is known to result in an increase in reactive oxygen species leading to an increase in the state of cellular oxidative stress. Consistent with its effects on cellular oxidative stress, exposure of cells that have been grown in media with physiologically normal glucose (5 mM) levels to higher levels of glucose (25 mM) under serum-free conditions results in a marked increase in cell death. This has been observed for several cell types including primary neuronal (1, 2), mesangial (3, 4), endothelial (5), and smooth muscle cells (SMCs) (6). In contrast to the response of cells to acute changes in glucose concentrations, when SMCs are grown, from the time of isolation, in medium containing 25 mM glucose and serum or transferred from 5 mM glucose to 25 mM glucose in the presence of serum, there is a minimal increase in cell death (7). Furthermore, when the chemotactic effect of serum or specific growth factors is examined in SMCs that have been adapted to 25 mM glucose, an increase in cell migration, compared with cells grown in 5 mM glucose, has been observed (8, 9). This enhanced response to growth factor stimulation occurs despite evidence of increased oxidative stress.

That diabetic patients have an increased risk of atherosclerotic vascular disease has been well documented; however, the reason for this risk is less well understood. The effect of glucose on SMC migration and proliferation, both of which are contributing factors to the development of atherosclerosis, has been studied by various researchers, but there is little knowledge as to the mechanism by which elevated glucose may alter the rate of SMC migration and proliferation. Whereas we and others routinely grow SMCs in medium containing 25 mM glucose, this is not physiologically normal. Understanding the components of growth factor signaling pathways that are activated when SMCs are grown in 25 mM glucose, compared with a more physiologically normal level of glucose, i.e. 5 mM, may provide insight into the mechanism by which hyperglycemia contributes to enhanced SMC migration and proliferation.

We have demonstrated that IGF-I can enhance the migration and proliferation of SMCs that have been isolated and maintained in media containing 25 mM glucose and serum (10). The ability of SMCs grown in 25 mM glucose to respond to IGF-I depends on the phosphorylation of the adaptor protein, Shc (11). We have shown that expressing a mutant form of Shc in which critical tyrosines are mutated to phenylalanine blocks the increase in cellular proliferation that occurs in response to IGF-I (11). The ability of IGF-I to stimulate Shc phosphorylation and thereby enhance SMC migration and proliferation is dependent on appropriate {alpha}Vß3 ligand occupancy, i.e. the addition of vitronectin (Vn) enhances IGF-I actions (12), whereas antibodies that block Vn binding to {alpha}Vß3 inhibit IGF-I actions (13). Prolonged hyperglycemia has been shown in various cell types including SMCs to increase production of various extracellular matrix (ECM) proteins that function as {alpha}Vß3 ligands including fibronectin (14, 15), osteopontin (Opn) (16), and thrombospondin-1 (TS-1). Given our previous data that demonstrate a role for {alpha}Vß3 ligand occupancy in regulating the response of SMCs to IGF-I, we hypothesized that increases in the level of ECM proteins that occur when SMCs are grown in hyperglycemic conditions enhance the response of SMCs to IGF-I.

The aim of this study was to compare the signaling, growth, and migration responses of SMCs maintained in medium containing a physiologically normal level of glucose (5 mM) with SMCs maintained in 25 mM glucose. In particular, we compared the ability of IGF-I to stimulate Shc phosphorylation, which is required for MAPK activation, which in turn is required for both migration and proliferation of SMCs in response to IGF-I (11). We also determined whether increasing {alpha}Vß3 ligand occupancy of SMCs maintained in normal glucose would increase the responsiveness to IGF-I to a level that is similar to the response when SMCs are maintained in high glucose.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human IGF-I was a gift from Genentech (South San Francisco, CA). Polyvinyl difluoride membranes (Immobilon P) were purchased from Millipore Corp. (Billerica, MA). Autoradiographic film was obtained from Pierce (Rockford, IL). Fetal bovine serum (FBS), DMEM, penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY). The IGF-I receptor (IGF-IR)-ß chain polyclonal and the monoclonal phosphotyrosine antibody PY99 and the insulin receptor substrate (IRS)-1 antibody were purchased from Santa Cruz (Santa Cruz, CA). The Shc, phospho/total ERK1/2, growth factor receptor-bound protein (Grb)-2, and anti-ß-actin antibodies were purchased from BD Transduction Laboratories (Lexington, KY). The polyclonal Vn antibody, the anti-SMC myosin heavy-chain antibody, and the monoclonal anti-SMC-{alpha} actin antibody were from Chemicon International (Temecula, CA). The polyclonal antibody to SM22-{alpha} was purchased from Abcam (Cambridge, MA). The polyclonal Opn antibody was raised in rabbits against the whole human Opn. The antibody used to detect ß3 protein by immunoblotting was generated in-house by injecting a rabbit with a peptide homologous to amino acids 36–63 of human ß3 conjugated to keyhole limpet hemocyanin. All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated.

Extracellular matrix proteins
Vn was purified from porcine serum as we have described previously (12). Opn was purified using a FLAG agarose affinity column from Chinese hamster ovary cells transfected with a pRCRSV construct containing cDNA encoding for porcine Opn with a FLAG tag. TS-1 was purchased from Chemicon.

Porcine SMCs
Porcine SMCs were isolated from the porcine aortic explants according to the protocol described by Ross (17). Briefly, after removal of interstitial tissue and endothelium, small sections of aortic explants were placed directly on the plastic surface of several 10-cm tissue culture dishes (catalog no. 3001; BD Falcon, Piscataway, NJ). The explants were covered with 10 ml of growth medium [DMEM containing 4500 mg/liter (25 mM) glucose plus 10% FBS and penicillin (1000 U/ml) and streptomycin (160 µg/ml)] referred to as high-glucose growth medium (HG-GM). SMCs were observed to have migrated from the explant after 4–7 d. Once SMCs were observed to have migrated from the explant and attached to the surface of the dish, the explants were removed (between 5 and 7 d). At this point, half of the plates were maintained in HG-GM, whereas the remaining half of the plates were maintained in DMEM containing 900 mg/liter of glucose (5 mM) plus 10% FBS and penicillin and streptomycin [normal-glucose growth medium (NG-GM)]. SMCs were fed every 3 d with either HG- or NG-GM and were passed every 7 d in appropriate medium. SMCs were maintained under these conditions for a further three passages before being used for experiments. All experiments were performed on SMCs between passage 4 and 10. Except where described, SMCs were maintained from passage to passage in medium with the same glucose concentration. Before initiation of each experiment, confluent monolayers were washed three times in serum-free medium (SFM) and incubated overnight (16–17 h) in SFM containing the glucose concentration equivalent to that in which they had been grown (25 mM glucose = SFM-H or 5 mM glucose = SFM-N). SFM-N was supplemented with 20 mM mannitol to ensure that any differences observed between the different glucose concentrations were not due to differences in osmolarity.

Cell migration
SMCs were seeded into each well of 6-well plates and grown in HG- or NG-GM. SMCs were fed once 3 d after seeding and then wounded 4 d later. Monolayers were wounded with a razor blade as we have described previously (10). After wounding SMCs were incubated with SFM-H or SFM-N plus 0.2% FBS in the presence or absence of IGF-I (100 ng/ml) at 37 C for 48 h. The wounded monolayers were then fixed and stained (Diff Quick; Dade Behring, Inc., Newark, DE), and the number of cells migrating into the wound area was counted. At least five of previously selected 1-mM areas at the edge of the wound were counted for each data point.

Cell proliferation
SMCs grown in HG- or NG-GM were plated at 2 x 104 cells/well in each well of a 24-well plate in SFM-H or SFM-N plus 2% FBS. Cells were left to attach overnight before the medium was replaced with SFM-H or SFM-N. 24 h later IGF-I (50 ng/ml) was added in SFM-H or SFM-N containing 0.2% platelet poor plasma and cells were incubated for a further 48 h. To examine the effect of Vn, Opn, or TS-1 after overnight incubation in SFM, one of each of the three proteins (Vn and Opn at 1 µg/ml and TS-1 at 0.5 µg/ml) was added alone or with IGF-I (50 ng/ml), and the incubation was continued for a further 48 h. Cell number was determined after trypsinization, trypan blue staining, and counting (12).

Measurement of protein synthesis
The measurement of protein synthesis was performed essentially as we described previously (18). SMCs were plated at 5 x 103 cells/well in HG- or NG-GM in each well of a 24-well plate (catalog no. 3047; BD Falcon) and maintained for 3 d to achieve 80% confluency. The media were removed and SMCs were incubated in 1.0 ml of SFM-H or SFM-N in the presence of 50 µCi 35S methionine and IGF-I (100 ng/ml) for 6 h. At the end of the incubation, the plates were placed on ice and washed with PBS before incubating with ice-cold trichloracetic acid (5%) for 10 min. After removal of the trichloracetic acid, the resulting precipitate was solubilized by incubating with 1% sodium dodecyl sulfate and 0.1 N NaOH overnight, and radioactivity was measured in a Beckman scintillation counter using Scintisafe Econo2 as a scintillant (Fisher Scientific, Fairlawn, NJ).

Membrane extraction protocol
SMCs that had been grown to confluency and exposed to SFM for 14 h were removed from the culture plate using a cell dissociation solution (catalog no. C5914; Sigma). Cell pellets were lysed by freezing and thawing. The membrane fraction was pelleted by centrifugation at 14,000 x g for 10 min and solubilized in 60 µl of solubilization buffer [150 mM NaCl, 50 mM HEPES (pH 7.4), 1% Nonidet P-40, and protease inhibitor cocktail III (Calbiochem/EMD Biosciences, La Jolla, CA)]. After a final centrifugation step (14,000 x g for 10 min), 30 µl of supernatant were separated by nonreducing SDS-PAGE, and proteins were visualized using Western immunoblotting.

Cell lysis, immunoprecipitation, and Western immunoblotting
SMCs were plated in 10-cm dishes in HG- or NG-GM and grown to confluency over 7 d with the medium being refreshed after 3 d. On d 7 the growth medium was removed and the confluent monolayers were rinsed three times with SFM-H or -N and then incubated overnight (16–17 h) in SFM before the addition of IGF-I (100 ng/ml) for the times indicated. Where indicated cell monolayers were treated with Vn (1 µg/ml), Opn (1 µg/ml), or TS-1 (0.5 µg/ml) for 2 h before the addition of IGF-I. Subsequent cell lysis, immunoprecipitation, and immunoblotting were performed as we previously described (19). For immunoblotting, antibodies were used at the following dilution: IRS-1, Shc, monoclonal phosphotyrosine antibody PY99, Grb-2, ß3, pERK1/2, ERK1, SMC myosin heavy chain, or SM22{alpha} at a 1:500 dilution and TS-1, Opn, Vn, or SMC{alpha} actin at a 1:1000 dilution.

Visualization of actin cytoskeleton
SMCs maintained in HG- or NG-GM were plated in the wells of a four-well chamber slide (catalog no. 28354104; BD Falcon) and left to attach overnight. After fixing with formaldehyde (4%) and permeabilization with triton (0.2%), SMCs were stained with Alexa Fluor 568 Phalloidin (Molecular Probes, Eugene, OR). The actin cytoskeleton was then visualized, and images were collected with a DMIRB inverted microscope (Leica, Bannockburn, IL) and a black-and-white cooled digital camera. Images were then transferred to Adobe Photoshop Elements (version 2; San Jose, CA) to prepare the figures.

Measurement of glucose consumption
SMCs grown in HG- or NG-GM were incubated for 24 h in SFM-H or SFM-N. Five microliters of media were removed and the glucose level was measured using the Freestyle glucose monitoring system (Abbott Laboratories, Alameda, CA). Glucose consumption was calculated by the difference in glucose levels at time zero and after 24 h incubation.

Statistical analysis
The chemiluminescent images that were obtained were scanned using a DuoScan T1200 (AGFA, Brussels, Belgium), and the band intensities of the scanned images were analyzed using National Institutes of Health Image (version 1.61). The Student’s t test was used to compare differences between treatments. The results that are shown in all experiments are representative of at least three separate experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation and migration of SMCs in response to IGF-I are enhanced in the presence of 25 mM glucose, compared with 5 mM glucose
After the addition of IGF-I, SMCs that had been maintained in 25 mM glucose exhibited a significant 2.5 ± 0.3-fold (mean ± SEM, n = 3, P < 0.005) increase in cell number over a 48-h period (Fig. 1AGo). However, when SMCs that had been grown in 5 mM glucose were examined, there was no significant increase in cell number in response to IGF-I over the same period of time [1.2 ± 0.09-fold increase (mean ± SEM, n = 3, P = ns)]. Similarly IGF-I stimulated a significant 3.13 ± 0.12-fold increase in cell migration in the presence of 25 mM glucose (mean ± SEM, n = 3, P < 0.005), whereas cells grown in 5 mM glucose showed no significant increase [1.2 ± 0.11-fold increase (mean ± SEM, n = 3, P = ns)] (Fig. 1BGo).


Figure 1
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  		FIG. 1. Effects of glucose concentrations on IGF-I stimulated migration and proliferation. A, Cells (2 x 104) grown in either 5 or 25 mM glucose were plated in SFM + 2% FBS in each well of a 24-well plate before exposure to IGF-I (50 ng/ml) in DMEM + 0.2% platelet poor plasma. Forty-eight hours after the addition of IGF-I, cell number was determined by trypan blue staining and counting. ***, P < 0.005 when cell number in response to IGF-I in cells grown in 25 mM glucose is compared with the response of cells grown in 5 mM glucose. B, Cells were grown to confluency in either 5 or 25 mM glucose in 6-well dishes. After wounding of the monolayer with a razor blade, IGF-I in SFM + 0.2% FBS (100 ng/ml) was added and cells were allowed to migrate over the wound line for 48 h. The number of cells migrating past the wound line into at least five predetermined 1-mm2 areas was then counted. The results shown are the mean ± SEM of three independent experiments. ***, P < 0.005 when the number of cells migrating in response to IGF-I in cells grown in 25 mM glucose is compared with the response to IGF-I of cells grown in 5 mM glucose.

 
The lack of an increase in migration and proliferation of SMCs grown in 5 mM glucose in response to IGF-I is not due to complete nonresponsiveness of these cells to IGF-I because we observed a 2.8 ± 0.3-fold increase (mean ± SEM, n = 3) in protein synthesis in SMCs grown in 5 mM. This is comparable with the 2.25 ± 0.4-fold increase (mean ± SEM, n = 3) we have previously seen when SMCs were grown in 25 mM glucose (18).

Effect of changes in glucose concentrations on IGF-I-stimulated Shc phosphorylation
We previously reported that the increases in cell migration and proliferation in response to IGF-I seen in SMCs maintained in 25 mM glucose require IGF-I stimulated p52 Shc phosphorylation (11). To determine whether a change in Shc phosphorylation accounted for the loss of a proliferation response to IGF-I, Shc phosphorylation was analyzed using both conditions. SMCs grown in 5 mM glucose showed no change in Shc phosphorylation in response to IGF-I [0.99 ± 0.03-fold increase (mean ± SEM, n = 3)]. In contrast, cells grown in 25 mM glucose demonstrated a significant increase in p52 Shc phosphorylation in response to IGF-I [3.3 ± 0.47-fold increase after10 min of IGF-I treatment (mean ± SEM, n = 3, P < 0.005) (P < 0.005 when phosphorylation of Shc in cells grown in 25 mM glucose is compared with cells grown in 5 mM glucose) (Fig. 2AGo)]. Consistent with the difference in Shc phosphorylation, a significant increase in Grb-2 binding to Shc could be demonstrated only in cells grown in 25 mM glucose [6.7 ± 1.02-fold increase in Shc binding to Grb-2 in cells grown in 25 mM glucose, compared with a 0.95 ± 0.02-fold increase in cells grown in 5 mM glucose, (mean ± SEM, n = 3 P < 0.005)]. The difference in Shc phosphorylation was not attributable to differences in levels of p52 Shc protein (Fig. 2AGo, lower panel).


Figure 2
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  		FIG. 2. Effect of glucose concentrations on IGF-I-stimulated Shc, IGF-IR, and IRS-1 phosphorylation. Cells were grown to confluency in either 5 or 25 mM glucose before overnight incubation in SFM with either 25 or 5 mM glucose. IGF-I (100 ng/ml) was added for the lengths of times indicated. A, The extent of Shc phosphorylation was determined by immunoprecipitating (IP) cell lysates with an anti-Shc antibody and then immunoblotting (IB) with an antiphosphotyrosine antibody (p-Tyr). The extent of Grb-2 association with Shc was determined by reprobing immunoblots with an anti Grb-2 antibody. Membranes were then stripped and reprobed with an anti-Shc antibody to demonstrate that there was no difference in the amount of Shc that was precipitated in each sample that would account for the difference in Shc phosphorylation and the amount of Grb-2 detected. The graphs show the mean fold increase in Shc phosphorylation and Shc-Grb-2 binding (n = 3 independent experiments) after 10 min stimulation with IGF-I (***, P < 0.005 when the increase in response to IGF-I in cells grown in 25 mM glucose is compared with the response of cells grown in 5 mM glucose). B, The extent of IGF-IR phosphorylation was determined by immunoprecipitating cell lysates with an anti-IGF-IR antibody and then immunoblotting with an antiphosphotyrosine antibody (p-Tyr). Membranes were then stripped and reprobed with an anti-IGF-IR antibody to demonstrate that there was no difference in the amount of IGF-IR protein that was precipitated in each sample. The graph shows the mean fold increase in IGF-IR phosphorylation (n = 3 independent experiments) after 10 min stimulation with IGF-I (P = ns when IGF-IR phosphorylation in SMCs grown in 25 mM glucose is compared with the response of SMCs grown in 5 mM glucose). C, The extent of IRS-1 phosphorylation was determined by immunoprecipitating cell lysates with an anti-IRS-1 antibody and then immunoblotting with an antiphosphotyrosine antibody (p-Tyr). The extent of Grb-2 association with IRS-1 was determined by reprobing with an anti-Grb-2 antibody. The graphs show the mean fold increase in IRS-1 phosphorylation and IRS-1 Grb-2 binding (n = 3 independent experiments) after 5 min stimulation with IGF-I (**, P < 0.01 when the increase in response to IGF-I in cells grown in 25 mM glucose is compared with the response of cells grown in 5 mM glucose). The control represents an immunoblot for ß3.

 
To demonstrate that the lack of Shc phosphorylation in SMCs grown in 5 mM glucose was not due to a complete lack of responsiveness to IGF-I, we compared IGF-IR phosphorylation at 5 and 25 mM. There was no significant difference in the extent of IGF-IR phosphorylation between the cells grown under the two different glucose conditions (Fig. 2BGo). The fold increases in IGF-IR phosphorylation for 25 and 5 mM glucose after 10 min treatment were 18 ± 0.46 and 15 ± 7.7, respectively (mean ± SEM, n = 3 P = ns).

As a further control, we compared the tyrosine phosphorylation response of IRS-1 after IGF-I stimulation under the two different glucose conditions. IRS-1 phosphorylation was significantly stimulated in cells grown in 5 mM glucose [3.4 ± 0.7-fold increase after 5 min stimulation with IGF-I (mean ± SEM, n = 3, P < 0.01)]. In cells grown in 25 mM glucose, the phosphorylation of IRS-1 was increased by only 1.25 ± 0.05 (mean ± SEM, n = 3, p = ns) (Fig. 2CGo). The difference in IRS-1 phosphorylation was also reflected by a marked reduction in Grb-2 binding to IRS-1 [1.9 ± 0.12-fold increase in cells grown in 5 mM glucose, compared with a 0.9 ± 0.03-fold increase in cells grown in 25 mM glucose (mean ± SEM, n = 3, P < 0.01 when the increase in the presence of 25 mM glucose is compared with the increase in 5 mM glucose) (Fig. 2CGo)].

Adaptation from 25 to 5 mM glucose
SMCs grown in culture undergo partial dedifferentiation acquiring a more motile and proliferative phenotype resembling the SMCs that contribute to the development of atherosclerosis, thereby providing a model for this disease (20). We determined previously that isolation of SMCs from vessel explants in 25 mM glucose yields SMCs that are partially dedifferentiated (21). To control for possible differences in differentiation state between SMCs grown in 5 and 25 mM glucose, we compared the levels of several markers of the SMC differentiation state in cells that had been maintained in medium with either 5 or 25 mM glucose, namely SMC actin, SM myosin heavy chain, and SM22 (22, 23, 24). In Fig. 3AGo, it can be seen that after immunoblot analysis of equal amounts of cell lysate, there was no significant difference in the level of each of the three markers of differentiation, SMC actin, SM myosin heavy chain, and SM22{alpha} between SMCs maintained in low or high glucose. Furthermore, there were no apparent differences in morphology after staining of the actin cystoskeleton (Fig. 3BGo). This is a cellular characteristic that has been shown to correlate with the state of differentiation (21, 23, 24, 25).


Figure 3
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  		FIG. 3. Comparison of differentiation state and adaptability of SMCs grown in 5 and 25 mM glucose. A, Markers of SMC differentiation were visualized after immunoblotting (IB) of cell lysates from SMCs grown in 5 or 25 mM glucose with appropriate antibody. B, SMCs grown in either 5 or 25 mM were fixed and stained with Alexa Fluor 568 phalloidin to visualize the actin cytoskeleton. C, SMCs isolated and maintained in 25 mM glucose were grown in medium containing 25 mM glucose or 5 mM glucose for 72 h before overnight incubation in SFM with either 25 or 5 mM glucose. IGF-I (100 ng/ml) was added for the lengths of times indicated. The extent of Shc phosphorylation was determined by immunoprecipitating (IP) cell lysates with an anti-Shc antibody then immunoblotting with an antiphosphotyrosine antibody (p-Tyr). To control for total amount of Shc protein, the blot was reprobed with an anti-Shc antibody The graph shows the mean fold increase in Shc phosphorylation (n = 2 independent experiments) after 5 min stimulation with IGF-I (**, P < 0.01 when the increase in response to IGF-I in cells grown in 25 mM glucose is compared with the response of cells that had been maintained for 72 h in 5 mM glucose).

 
To address the possibility that the difference in response to IGF-I between SMCs grown in 5 and 25 mM glucose might reflect selection of two different pools during isolation and culture, SMCs that had been isolated and maintained in 25 mM glucose were either maintained in 25 mM glucose or switched into medium containing 5 mM glucose for 72 h, and the ability of IGF-I to stimulate Shc phosphorylation was compared. Whereas IGF-I stimulated a significant increase (2.1 ± 0.5-fold mean ± SEM n = 2, P < 0.05) in Shc phosphorylation in SMCs maintained in 25 mM glucose, it did not stimulate a significant increase in Shc phosphorylation in the SMCs from the same population of cells that had been transferred to medium containing 5 mM glucose for 72 h (0.94 ± 0.05-fold increase) (Fig. 3CGo). This demonstrates that the difference in response to IGF-I between cells grown in 5 and 25 mM glucose is due to glucose concentrations and not to selection of two distinct populations of SMCs.

To ensure that the difference in response of SMCs grown in 5 and 25 mM glucose was not due to a difference in glucose consumption and/or the development of glucose depletion, the glucose concentrations were determined. There was no significant difference in the glucose consumption between the two culture conditions. There was a 1.8 ± 0.7 mM decrease in the levels of glucose in the medium from SMCs incubated with 25 mM glucose SFM (mean ± SEM, n = 3), compared with a 1.3 ± 0.2 mM decrease (mean ± SEM, n = 3) in the levels of glucose in the medium from SMCs incubated with 5 mM glucose after 24 h of incubation.

Effect of glucose on IGF-I stimulated downstream signaling pathways
We have previously shown that phosphorylation of Shc is required for IGF-I-stimulated MAPK activation and that MAPK activation is required for both migration and proliferation of SMCs in response to IGF-I (26). We therefore examined the phosphorylation of threonine 202 and tyrosine 204 of ERK as markers of activation of the MAPK pathway. IGF-I stimulated a significantly greater increase in ERK 1 rather than ERK 2 phosphorylation in cells grown in 25 mM glucose (Fig. 4Go). More specifically, the IGF-I stimulated increase in ERK1 phosphorylation in SMCs grown in 25 mM glucose was 18.3 ± 1.5- and 19 ± 5.5-fold greater than the response of SMCs grown in 5 mM glucose after a 10- and 20-min stimulation with IGF-I, respectively (mean ± SEM, n = 3, P < 0.005). These differences were not attributable to differences in the protein levels of ERK1.


Figure 4
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  		FIG. 4. The effect of glucose on IGF-I stimulated MAPK activation. Cells were grown to confluency in either 5 or 25 mM glucose before overnight incubation in SFM with either 5 or 25 mM glucose. IGF-I (100 ng/ml) was added for the times indicated. After cell lysis, aliquots containing equal amounts of cell lysate were separated directly by SDS-PAGE and immunoblotted (IB) with the antibodies indicated. Cell lysates were immunoblotted with antibody specific for phosphorylation of threonine 202 and tyrosine 204 of ERK (upper panel) and total ERK1 (lower panel). The graph shows the mean fold difference in ERK1 phosphorylation in cells grown in 25 mM glucose, compared with cells grown in 5 mM glucose, after 10 and 20 min stimulation with IGF-I (n = 3 independent experiments) (***, P < 0.005 when the increase in response to IGF-I in cells grown in 25 mM glucose is compared with the response of cells grown in 5 mM glucose).

 
Effect of {alpha}Vß3 ligands on IGF-I stimulated Shc phosphorylation and ERK activation in SMCs grown in 5 mM glucose
We have shown that in SMCs maintained in 25 mM glucose, Shc phosphorylation in response to IGF-I requires {alpha}Vß3 ligand occupancy (12) and that blocking ligand binding to {alpha}Vß3 with a ß3 antibody completely blocks the IGF-I stimulated increase in Shc phosphorylation (13). To determine whether the differences noted in SMC responsiveness might be due to differences in {alpha}Vß3 ligand occupancy, we compared the cell membrane-associated levels of three major {alpha}Vß3 ligands, TS-1, Opn, and Vn between cells grown in 5 and 25 mM glucose (Fig. 5AGo). We detected a 4.2 ± 1.3-fold increase in membrane-associated TS-1, a 3.7 ± 0.3-fold increase in Opn, and a 2.1 ± 0.32-fold increase in Vn (mean ± SEM, n = 3, P < 0.05 for each experiment) in SMCs grown in 25 mM, compared with 5 mM glucose. In all cases equal amounts of total cellular protein were analyzed to ensure that the differences in these three proteins did not reflect differences in the level of total cellular protein content.


Figure 5
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  		FIG. 5. Effect of glucose on cell membrane-associated {alpha}Vß3 ligands. A, Membrane extracts were prepared from cells grown in medium containing either 5 or 25 mM glucose. After separation by SDS-PAGE, proteins were visualized by immunoblotting (IB) with the appropriate antibody (TS-1, Opn, and Vn). To control for total protein, membrane extracts were also immunoblotted with an antibody to the cytoplasmic domain of ß3 subunit of {alpha}Vß3. The graph shows the mean fold increase in each of the proteins from cells grown in 25 mM glucose, compared with cells grown in 5 mM glucose (n = 3 independent experiments; ***, P < 0.005; **, P < 0.01). B, Cells were grown to confluency in 5 mM glucose before overnight incubation in SFM. After a 2-h incubation with Vn (1 µg/ml), Opn (1 µg/ml), or TS-1 (0.5 µg/ml), IGF-I (100 ng/ml) was added for the times indicated. Shc phosphorylation was determined by after lysis and immunoprecipitation (IP) with an anti-Shc antibody and immunoblotting with an antiphosphotyrosine antibody (p-Tyr). Blots were probed with an anti-Shc antibody to determine that differences in Shc phosphorylation were not due to differences in total amount of Shc protein. The graph shows the data expressed as arbitrary scan units, reflecting the extent of Shc phosphorylation after 5 min IGF-I stimulation (n = 2 independent experiments; ***, P < 0.005 when Shc phosphorylation in the presence of {alpha}Vß3 ligands is compared with Shc phosphorylation in their absence). C, Cells were grown and treated as above. After lysis, proteins were separated by SDS-PAGE before immunoblotting (IB) with an antibody specific for phosphorylated ERK1/2 or an antibody for total ERK1/2. The graph shows the data expressed as arbitrary scan units, reflecting the extent of ERK1 phosphorylation after 10 min of IGF-I stimulation (n = 2 independent experiments; ***, P < 0.005 when ERK phosphorylation in the presence of {alpha}Vß3 ligands is compared with ERK phosphorylation in their absence).

 
We hypothesized that if the increase in IGF-I responsiveness in SMCs grown in 25 mM glucose was attributable to changes in ligand binding to {alpha}Vß3, then addition of each of the {alpha}Vß3 ligands should alter the Shc phosphorylation and downstream signaling responses to IGF-I.

Addition of Vn, Opn, and TS-1 significantly increased the ability of IGF-I to stimulate Shc phosphorylation in normal glucose (Fig. 5BGo). Densitometric analysis (expressed as arbitrary scan units) demonstrated that the intensity of Shc phosphorylation, after 5 min stimulation with IGF-I was significantly increased from 199 ± 4.1 arbitrary scan units in SFM alone to 5091 ± 127, 15063 ± 350, and 16116 ± 479 arbitrary scan units (mean ± SEM, n = 2, P < 0.005) in the presence of Vn, Opn, and TS-1, respectively (Fig. 5BGo). Consistent with an increase in Shc phosphorylation, there were significant, 4.4 ± 0.2-, 5.4 ± 1.3-, and 11 ± 4-fold increases (mean ± SEM, n = 2, P < 0.005) in MAPK activation after 10 min stimulation with IGF-I in the presence of Vn, Opn, and TS-1, respectively (Fig. 5CGo). The consequence of the increased Shc and MAPK activation in response to exposure of SMCs to Vn, Opn, and TS-1 is shown in Table 1Go. The results show that in the presence of Vn, Opn, or TS-1, IGF-I was able to stimulate significant increases in SMC proliferation, even though the cells had been maintained in 5 mM glucose [1.9 ± 0.17-, 1.8 ± 0.12-, and 2.8 ± 0.8-fold increase in cell number in response to IGF-I, respectively (mean ± SEM, n = 3, P < 0.05 when the response to IGF-I in the presence of each {alpha}Vß3 ligand is compared with response to IGF-I in the absence of any added ligand)].


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  		TABLE 1. Cell proliferation in response to IGF-I in the presence or absence of the Vn, Opn, or TS-1 measured in SMCs grown in 5 mM glucose

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The goals of this study were to determine whether SMCs maintained in 5 mM (normal) or 25 mM (hyperglycemic) glucose differed in their response to IGF-I and to determine the mechanism by which cellular responsiveness was altered. Our previous studies have shown that Shc phosphorylation is required for MAPK activation in SMCs, which in turn is required for IGF-I to stimulate migration and proliferation (11). In this study, we show that there is a direct relationship between the glucose levels to which the cells are exposed and the ability of IGF-I to stimulate Shc phosphorylation. The complete lack of IGF-I stimulated Shc phosphorylation and the associated reduction in IGF-I stimulated ERK1 phosphorylation that we observed when cells were exposed to 5 mM glucose is consistent with the inability of these cells to increase migration and proliferation in response to IGF-I. In contrast, when cells were maintained in 25 mM glucose the Shc and MAPK phosphorylation responses to IGF-I were significantly increased.

Our previous studies that were conducted using SMCs maintained in 25 mM glucose have shown that {alpha}Vß3 ligand occupancy is required for SMCs to respond maximally to IGF-I (10, 13, 18). The addition of {alpha}Vß3 ligands such as Vn and Opn enhances the response of SMCs to IGF-I, whereas blocking the binding of these ligands to {alpha}Vß3 inhibits these cellular responses (13). The {alpha}Vß3 ligand occupancy regulates ß3 subunit phosphorylation (27). This increase in ß3 phosphorylation is required for Shc recruitment to the plasma membrane protein SHP-substrate-1 (SHPS-1) and for subsequent Shc phosphorylation (11). Therefore, ß3 ligand occupancy and phosphorylation regulate downstream signaling in response to IGF-I (27). Blocking {alpha}Vß3 ligand occupancy inhibits Shc phosphorylation and downstream signaling, whereas stimulating ß3 ligand occupancy enhances Shc phosphorylation and activation of the MAPK pathway (13).

In this study, we showed that cells grown in 25 mM glucose had significant increases in the amount of cell membrane-associated {alpha}Vß3 ligands, and this change was associated with increased Shc phosphorylation. More importantly, SMCs maintained in 5 mM glucose that had low levels of membrane-associated {alpha}Vß3 ligands and no increase in Shc phosphorylation in response to IGF-I were fully responsive to IGF-I in the presence of 5 mM glucose if {alpha}Vß3 ligand occupancy was increased by the addition of each of these {alpha}Vß3 ligands. Importantly, increasing {alpha}Vß3 ligand occupancy also restored the MAPK and growth responses to levels that were comparable with those seen in 25 mM glucose. Therefore, the major finding of this study is that exposure of SMCs to chronic hyperglycemic conditions results in enhanced production of ECM ligands that can bind to {alpha}Vß3. This allows IGF-I to phosphorylate Shc and activate MAPK, accounting for the enhanced proliferative response to IGF-I.

Glucose has been shown to up-regulate TS-1 (an {alpha}Vß3 ligand) gene expression, and the levels of TS-1 protein have been shown to be increased in response to glucose in other cell types such as mesangial cells (28, 29, 30). Furthermore, it has been shown that there are increased levels of TS-1 in the vessel walls of diabetic Zucker rats (31). Similarly, glucose has been shown to have a direct stimulatory effect on Opn production (32), and SMCs increase Opn secretion in response to hyperglycemia (33). Whereas there are no previous reports demonstrating a direct effect of glucose on Vn production, it has been reported that the levels of Vn are increased in renal tissue from streptozotocin-treated rats (34). Therefore, several prior studies support the concept that increased glucose concentrations stimulate an increase in the accumulation of {alpha}Vß3 ligands. Our studies extend these prior findings to show that the abundance of all three proteins is increased by glucose in cultured SMCs and that this increase results in major changes in IGF-I signaling as well as IGF-I-stimulated cell proliferation and migration. It is interesting to note that TS-1 appeared to be more effective than the other two ligands. Because TS-1 also binds several other cell surface proteins such as integrin-associated protein, which we have shown previously regulates IGF-I signaling (19), it is possible that TS-1 is acting through more than one mechanism to increase IGF-I signaling.

Because glucose consumption was the same in SMCs grown in 5 and 25 mM glucose, it suggests that the change in protein levels was not due to changes in the rates of cellular glucose metabolism. Culturing of cells for prolonged periods of time will result in the formation of advanced glycosylation end products (AGEs) (35). Several previous reports have demonstrated that AGEs enhance synthesis of TS-1 by various renal cells (36, 37). Similarly, the synthesis of Opn by pericytes (cells of the same lineage as smooth muscle cells) has been shown to be enhanced in the presence of AGEs (38). It has also been shown that modification of Vn by nonenzymatic glycosylation alters its functional properties (39). It is possible therefore that, although we did not detect as great a difference in Vn as with the other ECM proteins, its functional properties after exposure to high glucose are different. Furthermore, the relative contribution of each of these proteins to the SMC response to IGF-I when all three are present remains to be determined.

During the development of atherosclerosis, SMCs undergo partial dedifferentiation, allowing them to acquire a more motile and proliferative phenotype (40). Whereas it has been shown that SMCs in culture can be maintained in a differentiated state in vitro, this requires that these cells be plated on the specific ECM components, e.g. laminin and type IV collagen (21, 41, 42). The primary cultures of SMCs used in this study were isolated from smooth muscle explants using plastic tissue culture plates and cell culture medium containing FBS. SMCs cultured using these conditions have been reported to maintain a consistent state of partial dedifferentiation (21). Based on the lack of a significant difference in actin cytoskeleton staining and assessment of SMC differentiation markers, we conclude that the differences in the response of cells maintained in medium containing either 5 or 25 mM glucose were not due to differences in the state of cellular differentiation. Whether changes in glucose concentrations could alter the response of fully differentiated SMCs was not determined.

Because the effect of high glucose was reversed after 72 h incubation of SMCs in 5 mM glucose and the effect normal glucose was reversed by incubation with Vn, Opn, or TS-1, this suggests that the difference in response of SMCs grown in 5 and 25 mM glucose is not due to selection of different cell populations during the initial isolation or subsequent culture but rather to the glucose levels that directly influence the response of SMCs to IGF-I.

In a recent study, Hayashi et al. (43) showed that IRS-1 phosphorylation in response to IGF-I was required to stimulate cell migration and proliferation of dedifferentiated SMCs. IRS-1 phosphorylation in response to IGF-I activated the Ras/MAPK pathway, and this was required for the effects of IGF-I on migration and proliferation. Whereas the requirement of MAPK for IGF-I stimulated migration and proliferation is consistent with our findings, the role of IRS-1 in mediating the activation of MAPK is inconsistent with our results. It is difficult to make a direct comparison between our observations and those of Hayashi et al. because the glucose level of the medium in which the cells were maintained was not stated (43). Regardless of potential differences in glucose concentration, one important difference between the study of Hayashi et al. (43) and our study is the species from which the SMCs were derived. Hayashi et al. used SMCs isolated from rat aorta, whereas we used SMCs isolated from porcine aorta. This difference is significant due to the fact that the two amino acids that we have shown to be critical for the proper functioning of the cysteine loop region of ß3 (15) are not conserved between the rat (CYTMKSTC) and pig (CYDMKTTC) cysteine loop region of ß3. Because we have shown that ligand binding to this region of ß3 regulates IGF-I signaling (15), the lack of sequence similarity in this region of the rat ß3 subunit, which attenuates binding of Vn heparin binding domain to this integrin, may explain the difference in signaling response. It is important to note, that unlike the rat ß3 sequence, the amino acid sequence in the cysteine loop region of porcine ß3 is identical with the human ß3.

Our results show that SMCs that are grown in high glucose in the presence of serum migrate and proliferate in response to IGF-I. These responses are associated with enhanced Shc phosphorylation and MAPK activation in response to IGF-I. Whereas we cannot rule out the involvement of other signaling changes that might occur in response to hyperglycemia, given our previous data showing that the enhanced phosphorylation of Shc is required to enhance MAPK activation, we conclude that this change is required to detect subsequent changes in migration and proliferation. Consistent with our previous studies, which demonstrate a requirement for ligand occupancy of {alpha}Vß3 for SMCs to respond to IGF-I, these increases in the level of {alpha}Vß3 ligands appear to contribute to these enhanced responses of SMCs to IGF-I. SMCs maintained in 5 mM glucose are, however, not entirely nonresponsive to IGF-I, as demonstrated by the increases in IGF-IR and IRS-1 phosphorylation as well as protein synthesis. This is consistent with the blood vessel changes that have been reported in GH or IGF-I transgenic animals wherein the vessels exhibit hypertrophy but not hyperplasia (44). In contrast, the SMC hyperplastic response to IGF-I appears to require a stress signal. For example, an increase in IGF-I-stimulated SMC proliferation has been shown after mechanical injury (45) in the presence of hypercholesterolemia (46) or, as we have shown here, in the presence of hyperglycemia. This suggests that these stresses activate signaling pathways that are necessary for activated IGF-IR to induce SMCs to proliferate.


   Footnotes
 
This work was supported by National Institutes of Health Grant HL56850 (to D.R.C.) and American Heart Association Mid Atlantic Affiliate Beginning Grant in Aid 0465462U (to L.A.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 25, 2007

Abbreviations: AGE, Advanced glycosylation end product; ECM, extracellular matrix; FBS, fetal bovine serum; Grb, growth factor receptor-bound protein; HG-GM, high-glucose growth medium; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; NG-GM, normal-glucose growth medium; Opn, osteopontin; SFM, serum-free medium; SMC, smooth muscle cell; TS-1, thrombospondin-1; Vn, vitronectin.

Received October 27, 2006.

Accepted for publication January 17, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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