Increased Mitogenic Response to Heparin-Binding Epidermal Growth Factor–like Growth Factor in Vascular Smooth Muscle Cells of Diabetic Rats
Abstract We investigated the mitogenic effects of heparin-binding epidermal growth factor–like growth factor (HB-EGF) in vascular smooth muscle cells (SMCs) obtained from rats with streptozotocin (STZ)-induced diabetes and evaluated the role of heparan sulfate proteoglycan (HSPG) in inducing these effects. HB-EGF significantly increased DNA synthesis in the SMCs of diabetic rats (STZ-SMCs) compared with control rats (control SMCs). However, the mitogenic effects of EGF, which shares EGF receptors with HB-EGF, and basic fibroblast growth factor, another heparin-binding growth factor, were similar in STZ-SMCs and control SMCs. The mitogenic response to HB-EGF in SMCs of insulin-treated diabetic rats was similar to the response in control SMCs. HB-EGF–induced autophosphorylation of EGF receptors was increased in STZ-SMCs compared with control SMCs, although the number of EGF receptors in STZ-SMCs was 40% of that in controls. This increased mitogenic response to HB-EGF in STZ-SMCs was completely inhibited by treatment with heparitinase, chlorate, and a synthetic peptide corresponding to the heparin-binding domain of HB-EGF. Compared with heparan sulfate isolated from control SMCs, heparan sulfate isolated from STZ-SMCs was of smaller molecular size and caused a greater mitogenic effect of HB-EGF. These findings suggest that the mitogenic response to HB-EGF is increased in SMCs of diabetic rats. Changes in cell-associated heparan sulfate in STZ-SMCs may be related to the increased mitogenic response to HB-EGF.
- Received May 2, 1995.
- Accepted July 19, 1995.
Diabetes mellitus is a major risk factor for atherosclerosis.1 2 3 Hyperglycemia,3 hyperlipidemia,4 hyperinsulinemia,5 enhanced platelet aggregation,6 and elevated concentrations of lipid peroxides7 are all believed to contribute to diabetic macroangiopathy. Because SMC proliferation and migration are essential components of these processes,8 a fundamental understanding of the mechanisms of SMC proliferation and migration would greatly facilitate the development of effective strategies for preventing diabetes-induced atherosclerosis. Although cultured aortic SMCs from STZ-induced diabetic rats and alloxan-induced diabetic rabbits have exhibited faster growth than normal SMCs,9 10 11 the biologic mechanisms of the growth of SMCs in the diabetic state have not been clarified.
HB-EGF, a protein secreted by the human macrophage-like cell line U-937, is a potent mitogenic and migratory factor for SMCs.12 13 14 15 Mature bioactive HB-EGF is a 22-kD glycoprotein that binds to the EGF receptor, stimulating phosphorylation. The primary sequence of HB-EGF contains a COOH-terminal portion that is 40% structurally identical to EGF and to TGF-α and an NH2-terminal extension region that is not present in EGF or TGF-α.12 13 HB-EGF is synthesized by macrophages, monocytes,16 vascular endothelial cells,17 18 keratinocytes,19 and SMCs.20 Previously we have observed the increased expression of HB-EGF in SMCs and macrophages of human atherosclerotic plaques.21 Lysophosphatidylcholine, a major component of oxidized LDL, increases HB-EGF mRNA expression in human monocytes16 and vascular endothelial cells.17 Shear stress also upregulates the level of HB-EGF mRNA in human umbilical vein endothelial cells.18 Expression of HB-EGF mRNA in SMCs is induced by platelet-derived growth factor (PDGF), b-FGF, angiotensin II, and HB-EGF itself.20 These findings suggest that HB-EGF may have a regulatory role in the growth of SMCs in vivo.
HB-EGF demonstrates a more potent mitogenic effect on SMCs than does EGF and TGF-α, although all three growth factors bind to the EGF receptor.12 The heparin-binding properties of HB-EGF may facilitate its interaction with cell-surface HSPG, which may modulate its biologic activity.12 13 22 Such facilitation has been observed in the case of other heparin-binding growth factors, such as b-FGF.
In the present study, we examined the mitogenic response of STZ-SMCs to HB-EGF in addition to the effect of HS synthesized by these SMCs on the mitogenic activity of HB-EGF.
Recombinant human HB-EGF was kindly provided by Dr Judith A. Abraham, Scios Nova Inc.23 Recombinant human EGF was purchased from Toyobo Co, Ltd. Recombinant human b-FGF was purchased from Austral Biologicals. The 125I-EGF (3.7 MBq/mL) used in our experiments was purchased from Amersham Life Science.
Diabetes was induced in male Wistar rats (250 g) by administering intravenous injections of STZ (70 mg/kg) (Wako). Beginning 3 days after these injections, half the diabetic rats underwent a series of subcutaneous injections of protamine zinc insulin (Novo Nordisc) administered daily at 5 pm for 4 weeks. Age-matched control and diabetic rats were fed standard chow for 4 weeks. Fed rats were then anesthetized with sodium pentobarbital (30 mg/kg) and exsanguinated via the abdominal aorta. Serum and plasma samples were prepared from collected blood, and the plasma concentration of glucose and the serum concentrations of immunoreactive insulin, triglycerides, total cholesterol, and free fatty acids were measured by previously described methods.24 Diabetic rats were characterized by significant hyperglycemia, hypoinsulinemia, and low body weight. Serum free fatty acid, triglyceride, and total cholesterol concentrations were significantly elevated in diabetic rats compared with controls. Insulin treatment partially normalized some of these biochemical parameters (Table 1⇓).
Culture of SMCs
Rat aortic SMCs were isolated using the explant method of Fischer-Dzoga et al.25 In brief, aortic explants were obtained from the thoracic aorta and the adventitia was removed. Explants were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (Irvine Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO2. After 2 weeks, the cells that had migrated out of the explant were removed by trypsinization and seeded in T-75 flasks. Cells were characterized as SMCs by morphological criteria (spindle shape and hill-and-valley pattern) and by their expression of smooth muscle α-actin. SMCs were subcultured at a 1:4 split ratio every week. Cells from the fourth to sixth passages were used in the following experiments. Three different strains of control SMCs and STZ-SMCs and two different strains of insulin-treated diabetic rat SMCs were used, with similar results.
Effects of Growth Factors on DNA Synthesis in SMCs
DNA synthesis in SMCs was assessed by [3H]thymidine incorporation. Cells were grown to subconfluence (≈30 000 to 40 000 cells/cm2) in 96-well plates and placed in a growth-arresting medium consisting of DMEM containing 0.1% bovine plasma-derived serum (0.1% PDS-DMEM) for 48 hours to induce a quiescent state. The medium was then replaced with 5% PDS-DMEM containing various concentrations of HB-EGF, EGF, or b-FGF, and the cells were cultured for 24 hours. PDS, which is devoid of platelet-derived mitogens, maintains SMCs in the quiescent state for extended periods of time in culture.26 Twenty hours after the addition of growth factors, cells were pulse labeled with [3H]thymidine (37 kBq/well) (Amersham) for 4 hours. Incorporated [3H]thymidine was counted via a beta plate system (Pharmacia LKB).
EGF Receptor Binding Assay
Cells were washed twice with a binding buffer (DMEM, 50 mmol/L BES, and 0.1% BSA) and incubated for 4 hours at 4°C in 24-well plates with various amounts of 125I-EGF (3.7 MBq/mL) in the presence or absence of a 100-fold excess of unlabeled EGF.27 The cells were then washed four times with the binding buffer and solubilized with 0.75 mL of a lysing buffer (0.01 mol/L Tris-HCl, 0.5% SDS, and 1 μmol/L EDTA). Cell-bound and free 125I radioactivity was measured with a gamma counter. Nonspecific binding to cells was usually ≈≤10% of the total binding. Data were applied to Scatchard plots using least-squares analysis.
Autophosphorylation of the EGF Receptor
Autophosphorylation of the EGF receptor was assessed according to the method of Margolis et al.28 Quiescent SMCs were stimulated with 100 ng/mL HB-EGF for 10 minutes at 37°C. These cells were scraped into a lysis buffer containing 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mmol/L PMSF, 200 μmol/L sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, and 30 mmol/L p-nitrophenyl phosphate. After the protein content was measured according to the method of Lowry et al,29 lysates of SMCs (100 μg protein) were immunoprecipitated with sheep anti-human EGF receptor antibody (No. 06-129) (UBI) coupled to protein G–Sepharose (Sigma). After immunoprecipitation, samples were washed with the lysis buffer, placed in the sample buffer (25 mmol/L Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; and 0.05% bromophenol blue), and heated to 90°C for 5 minutes. Immunoprecipitated proteins were then separated on an SDS-gel gradient (SPG-520L, ATTO) and transferred to polyvinylidene difluoride membranes (Immobilon PVDF, Millipore). Phosphorylation of EGF receptors on tyrosine residues was analyzed by Western blot analysis by using rabbit anti-phosphotyrosine antibodies (No. 05-321) (UBI) and subsequent development with peroxidase-conjugated swine anti-rabbit IgG antibodies (Dako). Visualization by fluorography with an enhanced chemiluminescence system (Amersham) was then performed.
Heparitinase and Chlorate Treatment of Cells
To investigate the relationship between HSPG and the biologic activity of HB-EGF, SMCs were treated as previously described with heparitinase to digest cell-surface HS or with chlorate, an inhibitor of HS sulfation.22 For heparitinase treatment, SMCs were preincubated with 20 mU/mL heparitinase I (EC 126.96.36.199) (Sigma), beginning 1 hour before the addition of HB-EGF or EGF. For chlorate treatment, SMCs were preincubated with 10 mmol/L sodium chlorate (Aldrich) beginning 48 hours before the addition of growth factors. The effects of growth factors on DNA synthesis were investigated as described above.
Treatment of SMCs With a Synthetic Peptide Corresponding to the Heparin-Binding Domain of HB-EGF
A synthetic peptide (P21) corresponding to the putative 21–amino acid heparin-binding sequence KRKKKGKGLGKKRDPCLRKYK of human HB-EGF (amino acids 93 through 113 of the 208-residue precursor)22 was synthesized by use of an Applied Biosystems Peptide Synthesizer (courtesy of Margaret Ehrhardt [Brigham and Women’s Hospital, Boston, Mass]). Cells were preincubated with 25 μg/mL P21 beginning 1 hour before the addition of HB-EGF and b-FGF. The DNA synthesis in SMCs was investigated as described above.
Isolation of Cell-Associated Protein-Free Glycosaminoglycans
Cell-associated GAGs were isolated according to the method of Schmidt and Buddecke.30 In brief, 10 quiescent cultures (10-cm dishes) of SMCs were preincubated at 37°C for 48 hours in the presence of [35S]sulfate (100 kBq/mL) (Amersham) or d-[1,6-3H(N)]glucosamine hydrochloride (93 kBq/mL) (New England Nuclear). Then the medium was removed, and the cell layer was washed twice with PBS and trypsinized. The trypsin digest was precipitated with 2.5-vol ethanol and potassium acetate. Next, the precipitate was digested with 5 mg of crystallized papain (Wako) in 50 mmol/L sodium acetate (pH 5.8), containing 10 mmol/L EDTA and 5 mmol/L cysteine, at 65°C for 24 hours. The resulting digest was centrifuged, and the supernatant was applied to a DEAE column (1 mL, DE 52, Whatman) equilibrated with 50 mmol/L Tris-HCl (pH 7.0), containing 1% Triton X-100 and preloaded with 5 mg BSA. The column was washed with 3 vol of 50 mmol/L Tris-HCl, containing 1% Triton X-100 and 3 vol of 50 mmol/L Tris-HCl, and then eluted in a stepwise manner with 3 mL each of 0.1-mol/L and 0.7-mol/L NaCl buffered with Tris-HCl. The 0.7-mol/L eluate (containing the total GAGs) was digested with 10 mU/mL chondroitin ABC lyase (Seikagaku) according to the method of Saito et al.31 The digest was again subjected to DEAE chromatography under identical conditions, and the HS fraction was obtained. Incorporation of 35S or 3H radioactivity in HS was determined by scintigraphy. Cell protein contents per dish were determined before trypsin digest.
Sephacryl S-200 Chromatography of HS
For determination of the relative molecular mass, isolated HS was subjected to chromatography using a Sephacryl S-200 (Pharmacia) column (1×50 cm) equilibrated with 50 mmol/L sodium acetate (pH 6.0), 50 mmol/L NaCl, and 0.2% SDS. After application of the sample, the column was eluted with the same buffer at a flow rate of 10 mL/h, and 0.5-mL fractions were collected. 35S radioactivity in each fraction was determined using a scintillation counter.
Effect of Cell-Associated HS on the Mitogenic Activity of HB-EGF
Nonradiolabeled HS was isolated from quiescent control SMCs (control-HS) and STZ-treated SMCs (STZ-HS). Control SMCs were treated with 5 mmol/L chlorate for 48 hours or with 20 mU/mL heparitinase for 4 hours. Various concentrations of HS were then coincubated with HB-EGF for 24 hours. DNA synthesis in SMCs was then determined.
Data are presented as mean±SD. Differences between group means were analyzed by the Student’s t test as indicated in the tables and figure legends. A value of P<.05 was considered statistically significant.
Effects of Growth Factors on Proliferation of SMCs
In concentrations of up to 2.5 nmol/L, HB-EGF stimulated DNA synthesis in a dose-dependent manner in control SMCs, STZ-SMCs, and SMCs of insulin-treated diabetic rats (insulin-treated STZ-SMCs) (Fig 1⇓). The mitogenic response to HB-EGF was significantly higher in STZ-SMCs than in control SMCs. Insulin treatment inhibited this increase in the mitogenic response in STZ-SMCs. The response to EGF was almost similar in all three groups (Fig 1⇓), and the mitogenic effect of HB-EGF in STZ-SMCs was significantly more potent than the effect of EGF. The mitogenic response to b-FGF was almost similar in all three groups (Fig 1⇓). We tested three growth factors at concentrations up to 10 nmol/L. In higher concentrations than 2.5 nmol/L, the response curve did not show dose dependence. Sometimes, the response at 10 nmol/L was lower than that at 2.5 nmol/L or 0.62 nmol/L (data not shown).
EGF Receptor Analysis
Scatchard analysis demonstrated that the affinity of EGF for EGF receptors was similar in control SMCs and STZ-SMCs (Kd, 320 pmol/L and 300 pmol/L, respectively). The number of receptors on the surfaces of STZ-SMCs was 40% of the number on control SMCs (1.26×104/cells versus 3.13×104/cells) (Fig 2⇓).
EGF Receptor Autophosphorylation
Western blot analysis demonstrated that HB-EGF–induced autophosphorylation of the EGF receptor was significantly increased in STZ-SMCs compared with control SMCs (Fig 3⇓).
Effects of Chlorate and Heparitinase on Stimulation of DNA Synthesis by HB-EGF and EGF
The inhibitory effect of 10 mmol/L chlorate on [3H]thymidine incorporation was significantly greater in HB-EGF–treated cells than in EGF-treated cells (Fig 4⇓). In addition, the inhibitory effect of chlorate was more pronounced in STZ-SMCs than in control SMCs (85% inhibition in STZ-SMCs versus 65% in control SMCs). Similarly, 20 mU/mL heparitinase caused greater inhibition of the mitogenic response to HB-EGF in STZ-SMCs than in control SMCs (70% in STZ-SMCs versus 50% in control SMCs) (Fig 5⇓). After chlorate and heparitinase treatment, the response to HB-EGF was similar in control SMCs and STZ-SMCs.
Effects of a Synthetic Peptide Corresponding to the Heparin-Binding Domain of HB-EGF on DNA Synthesis Stimulated by HB-EGF and b-FGF
The synthetic peptide P21 inhibited the mitogenic response to HB-EGF more potently in STZ-SMCs than in control SMCs (50% inhibition versus 20% inhibition) (Fig 6⇓). After P21 treatment, the response to HB-EGF was similar in STZ-SMCs and control SMCs. P21 did not alter the mitogenic activity of b-FGF.
Cell-Associated HS in Control SMCs and STZ-SMCs
Incorporation of 35S and 3H in HS was 70% lower in STZ-SMCs than in control SMCs. The 35S/3H ratio for HS was similar in control SMCs and STZ-SMCs (Table 2⇓). The partition coefficient (Kav) of elution maxima of HS from STZ-SMCs was higher than that of HS from control SMCs (Kav, 0.23 in STZ-SMCs versus Kav, 0.11 in control SMCs) (Fig 7⇓).
Effect of HS on Bioactivity of HB-EGF in SMCs
HS isolated from SMCs restored the mitogenic response to HB-EGF in SMCs treated with chlorate in a dose-dependent manner. STZ-HS was approximately twice as potent as control HS for equivalent amounts of cell protein (Fig 8⇓). STZ-HS also increased the response to HB-EGF in SMCs treated with heparitinase in the same manner (data not shown). HS did not affect the basal [3H]thymidine incorporation in quiescent SMCs.
In the present study, we demonstrate that the mitogenic effect of HB-EGF was significantly increased in STZ-SMCs compared with control SMCs. However, the mitogenic effects of EGF, which shares EGF receptors with HB-EGF, and b-FGF, another heparin-binding growth factor, were similar in STZ-SMCs and control SMCs. The mitogenic response to HB-EGF in SMCs of insulin-treated diabetic rats was similar to the response in control SMCs. HB-EGF is a potent mitogenic and migratory factor for SMCs that is equal to PDGF12 13 14 15 22 and is synthesized by macrophages, monocytes,16 vascular endothelial cells,17 18 and SMCs.20 Thus, an increase in the responsiveness to HB-EGF may be involved in the pathogenesis of diabetic vascular changes.
HB-EGF–induced autophosphorylation of EGF receptors was significantly increased in STZ-SMCs, although the number of EGF receptors was decreased to only 40% of the control number. This increased mitogenic response to HB-EGF in STZ-SMCs was completely inhibited by treatment with heparitinase, chlorate, and a synthetic peptide corresponding to the heparin-binding domain of HB-EGF. HS isolated from STZ-SMCs enhanced the bioactivity of HB-EGF to a greater degree than HS isolated from control SMCs. These findings suggest that the increased responsiveness to HB-EGF in STZ-SMCs may have been related to alterations of cell-surface HS chains rather than to changes in the EGF receptor itself.
Previous studies have shown that the biologic activities of b-FGF, vascular endothelial growth factor, and HB-EGF, which are all heparin-binding growth factors, depend on a dual receptor system consisting of a high-affinity receptor and a cell-surface-specific HSPG.32 33 34 35 Possible mechanisms of the activity of HSPG in the regulation of growth factor activity are (1) HSPG concentrates heparin-binding growth factors on the cell surface, making them more readily available to the cell,36 (2) HSPG stabilizes or alters the conformation of heparin-binding growth factors and/or their receptors, thereby increasing ligand-receptor affinity,34 35 (3) HSPG stabilizes dimerization of heparin-binding growth factors, which in turn facilitates receptor dimerization,37 38 and (4) HSPG lowers the off-rate component of binding to high-affinity receptors by heparin-binding growth factors.39
Several species of HSPG, such as syndecan, fibroglycan, and glypican, fail to promote high-affinity receptor binding of b-FGF, although total cell-derived HSPG promotes such binding.40 This fact suggests that a unique species of HSPG may be involved in the regulation of receptor binding and the biologic activity of b-FGF. A recent study has identified perlecan as a major candidate for a b-FGF low-affinity receptor.41 In this study, P21, a synthetic peptide corresponding to the heparin-binding domain of HB-EGF, inhibited HB-EGF–mediated effects, but not b-FGF–mediated effects, suggesting that the HSPG species that corresponds to the heparin-binding domain of HB-EGF may differ from the species that corresponds to the domain of b-FGF. Therefore, changes in HS of STZ-SMCs may selectively correspond to the heparin-binding domain of HB-EGF, thus stimulating the bioactivity of HB-EGF.
The amount of cell-associated HSPG determined by the incorporation of [35S]sulfate or [3H]glucosamine was significantly decreased in STZ-SMCs compared with control SMCs. In addition, the relative molecular mass of HS isolated from STZ-SMCs was smaller than that of HS isolated from control SMCs. These findings suggest that the HS in STZ-SMCs is qualitatively different from that in control SMCs. However, the mechanism whereby altered HS in STZ-SMCs potentiated the mitogenic effect of HB-EGF was not clarified in this study. It is not clear which species of HSPG stimulates ligand-receptor binding, thus regulating the bioactivity of HB-EGF. Further studies are needed to clarify the differences between cell-associated HS in control SMCs and STZ-SMCs and the detailed characterization of HS that mediates the bioactivity of HB-EGF.
The increased mitogenic effect of HB-EGF in SMCs of diabetic rats was normalized by insulin treatment, suggesting that the effect was caused by metabolic changes induced by an insulin deficiency. Several studies have shown that HSPG synthesis is altered in the diabetic state in various types of cells. Among the metabolic changes observed in diabetes, hyperglycemia has been thought to be a possible factor causing the alteration of HSPG synthesis. High doses of glucose have been found to alter proteoglycan synthesis in avian cartilage cells.42 High-dose glucose has also been observed to reduce HSPG synthesis in mesangial cells, but the mechanism of this effect remains unclear.43 44 In SMCs, HSPG synthesis is altered by cytokines such as tumor necrosis factor–α45 or by a proliferating state.30 However, HSPG synthesis in SMCs in the diabetic state have not been well examined. Therefore, we are currently investigating regulation of HSPG synthesis in SMCs by glucose and insulin.
The increased mitogenic response to HB-EGF in STZ-SMCs persisted during passages up to the sixth, indicating that the change was not a transient response to alterations in the levels of glucose, insulin, triglycerides, or other factors. Stability in the presence of passaging suggests a genetic difference between STZ-SMCs and control SMCs. We might have obtained a different subpopulation of SMCs from the STZ-treated and control rat aortas.
In this study, we observed no SMC hyperplasia or migration into the intima in our STZ-induced diabetic rats, which are common phenomena in atherosclerotic lesions in the human aorta.8 It remains to be clarified whether factors other than HB-EGF are required to induce such atherosclerotic lesions in STZ-treated rats or whether the absence of such lesions in the present study was related to the relatively short experimental period. Further study is needed to determine whether similar alterations in responsiveness to HB-EGF occur in SMCs derived from atherosclerotic lesions.
In conclusion, our results demonstrate that the mitogenic responsiveness to HB-EGF is significantly increased in STZ-SMCs, possibly because of changes in the cell-surface HS.
Selected Abbreviations and Acronyms
|b-FGF||=||basic fibroblast growth factor|
|control SMCs||=||SMCs derived from control rats|
|EGF||=||epidermal growth factor|
|HB-EGF||=||heparin-binding epidermal growth factor–like growth factor|
|HSPG||=||heparan sulfate proteoglycan|
|SMC(s)||=||vascular smooth muscle cell(s)|
|STZ-SMCs||=||SMCs derived from STZ-induced diabetic rats|
|TGF||=||transforming growth factor|
This work was supported in part by a grant-in-aid for cancer research to Drs Higashiyama and Taniguchi (No. 05151047), a grant-in-aid to Dr Matsuzawa (No. 04404085), and a grant-in-aid to Dr Kawata (No. 06557058) from the Ministry of Education, Science, and Culture of Japan. Dr Higashiyama is the recipient of a Searl Scientific Research Fellowship and a Suzuken Memorial Foundation Fellowship. We are grateful to Dr Judith A. Abraham for her generous gift of recombinant human HB-EGF.
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