Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells

Atherosclerosis 157 (2001) 341–352

Glucosamine enhances platelet-derived growth factor-inducedDNA synthesis via phosphatidylinositol 3-kinase pathway in rat

aortic smooth muscle cells

Akira Sato a, Toshiyasu Sasaoka b,*, Katsuya Yamazaki a, Norio Nakamura a,Rie Temaru a, Manabu Ishiki a, Michiyo Takata a, Mika Kishida a, Tsutomu Wada a,

Hajime Ishihara a, Isao Usui a, Masaharu Urakaze a, Masashi Kobayashi a

a First Department of Medicine, Toyama Medical and Pharmaceutical Uni�ersity, Toyama, Japanb Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical Uni�ersity, Toyama, Japan

Received 5 April 2000; received in revised form 3 November 2000; accepted 9 November 2000

Abstract

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cellswith high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long periodof high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosaminebiosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culturemedia enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF �-receptorwas increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF �-receptorassociation with PLC� was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, andMAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF �-receptor association with the p85regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P�0.01) and 36% (P�0.01), respectively. Theintracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors.Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitoryeffect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantlyinhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involvedin the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway. © 2001Elsevier Science Ireland Ltd. All rights reserved.

Keywords: PDGF; DNA synthesis; Glucosamine; Atherosclerosis; Vascular smooth muscle cell

www.elsevier.com/locate/atherosclerosis

1. Introduction

Diabetes Mellitus is one of the major risk factors foratherosclerosis. The prevalence of atherosclerotic vascu-lar disease in diabetic patients is two- to threefoldgreater than that in non diabetic subjects [1]. Althoughthe precise mechanisms of progression of atherosclero-sis in diabetes are uncertain, vascular smooth musclecells (VSMCs) appear to play a key role in the develop-

ment of atherosclerosis [2]. Platelet-derived growth fac-tor (PDGF) is one of the principal regulators ofmitogenesis in VSMCs [2], since expression of PDGF isincreased in atherosclerotic lesions [3] and PDGF-in-duced mitogenesis and migration are shown to be pre-requisites for intimal thickening after angioplasty [4–6].Therefore, elucidation of the regulatory mechanism ofPDGF signaling in the diabetic state is important forunderstanding the pathogenesis of atherosclerosis indiabetes. PDGF binding to the PDGF �-receptor leadsto its phosphorylation on multiple tyrosine residues [7].The activated PDGF �-receptor associates with a num-ber of SH2 domain-containing proteins including the

* Corresponding author. Tel.: +8l-76-4347287; Fax: +8l-76-4345025.

E-mail address: [email protected] (T. Sasaoka).

0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.PII: S 0 0 2 1 -9150 (00 )00740 -1

A. Sato et al. / Atherosclerosis 157 (2001) 341–352342

p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, phospholipase C (PLC)-�, phosphotyrosinephosphatase SHP2 (previously called Syp, SH-PTP2, orPTP-1D), GTPase activating protein of p21ras (GAP),Src, Nck, Grb2, and Shc [7]. Of these downstreammolecules of the PDGF �-receptor, involvement of Shc,Grb2, PLC�, and PI3-kinase in PDGF-induced mitoge-nesis has been relatively well characterized [8–10].Thus, PDGF-stimulated tyrosine phosphorylation ofShc functions as a predominant docking protein toGrb2 resulting in Ras-MAP kinase activation and mito-genesis in rat A10 aortic smooth muscle cells [11,12].PLC� and PI3-kinase pathways also have been shownto be important for PDGF-induced cell cycle progres-sion in VSMCs [13–16]. Although increasing evidenceexists supporting an important role of PDGF-inducedmitogenesis in VSMCs in the development and progres-sion of atherosclerosis in diabetes [2–6], the exactmechanism of modulation of PDGF-induced mitogene-sis in the diabetic state through these intracellular sig-naling pathways is unknown.

Flux of excess glucose to the hexosamine biosyntheticpathway is one of major causes of insulin resistanceelicited by hyperglycemia in insulin’s target tissues [17].The addition of glucosamine, a product of thehexosamine biosynthetic pathway, or stimulation of thehexosamine biosynthetic pathway by overexpression ofglutamine:fructose-6-phosphate amido-transferase(GFAT), which is the rate-limiting enzyme inhexosamine synthesis, resulted in insulin resistance [18–26]. Thus, the role of the hexosamine biosyntheticpathway in causing insulin resistance is well character-ized in insulin’s target tissues. Glucosamine is alsoknown to be a transcriptional stimulator of transform-ing growth factor-� (TGF-�), transforming growth fac-tor-� (TGF-�) and fibroblast growth factor (FGF)-2 insome VSMCs [27–30]. Although previous reports indi-cate a role of glucosamine in certain aspects ofatherosclerosis [27–30], the effect of glucosamine on theproliferation of VSMCs is uncertain.

In this study, we used rat aorta-derived smoothmuscle A10 cells, since PDGF signaling and cell growthare relatively well characterized in these cells as a modelof VSMC [12,31]. We demonstrated that glucosamineenhanced PDGF-induced DNA synthesis in A10 cells.Intracellular signaling pathways responsible for glu-cosamine enhancement of PDGF-induced DNA synthe-sis were investigated and further characterized usingpharmacological inhibitors.

2. Materials and methods

2.1. Materials

A polyclonal anti-PDGF �-receptor antibody was

purchased from Santa Cruz Biotechnology (Santa Cruz,CA). A monoclonal anti-Grb2 antibody, a monoclonalanti-phosphotyrosine antibody (pY20), and a poly-clonal anti-Shc antibody were from Transduction Lab-oratories (Lexington, KY). A polyclonal anti-p85subunit of PI3-kinase antibody and a monoclonal anti-PLC�-1 antibody were from Upstate Biotechnology(Lake Placid, NY). A polyclonal anti-phospho-specificp44/42 MAP kinase antibody and MEK1 inhibitor,PD98059, were from New England Biolabs (Beverly,MA). PLC inhibitor, U73122, was from BIOMOL Re-search Laboratories (Plymouth Meeting, PA). Humanrecombinant PDGF-BB was from Life Technologies(Grand Island, NY). [�-32P]ATP (220 Bq/mmol) wasfrom Amersham Pharmacia Biotech (Uppsala, Swe-den). PI3-kinase inhibitor (LY294002), azaserine, andthe other routine reagents were of analytical grade andpurchased from Sigma Chemical Co. (St. Louis, MO)or Wako Pure Chemical Industries, Ltd. (Osaka,Japan).

2.2. Cell culture and treatment

A10 VSMCs derived from rat thoracic aorta wereobtained from American type culture collection. Cellsat the passages of 9 to 13 were used for studies, and thephenotype of the cells was not changed at these pas-sages. The cells in 10 cm dish were maintained inDMEM containing 5.5 mM glucose, 10% FCS, 1.25mg/ml fungizone, 100 U/ml penicillin, and 100 �g/mlstreptomycin. The cells were further cultured in thecontrol medium, in the high glucose medium containing20 mM glucose, or in the medium supplemented with14.5 mM mannitol for 4 weeks. The culture mediumwas replaced every two days and the cells were pas-saged every week. In the last passage, same numbers ofthe cells were subcultured, and the cells that becamefully confluent were starved for 24 h in DMEM con-taining 1% FCS and the indicated concentrations ofglucose for the experimental usage. For the cell culturewith glucosamine, cells were cultured in the controlmedium supplemented with indicated concentrations ofglucosamine for 1 week. The culture medium was alsoreplaced every two days. The confluent cells werestarved for 24 h in DMEM containing 1% FCS andindicated concentrations of glucosamine, and were usedfor following studies. For the study with various in-hibitors, indicated concentrations of MEK1, PI3-ki-nase, or PLC inhibitors were added 1 h before PDGFstimulation.

2.3. Western blotting

Serum-starved A10 cells in 10 cm dishes were stimu-

A. Sato et al. / Atherosclerosis 157 (2001) 341–352 343

lated with 167 pM PDGF at 37°C for the indicatedtimes. The cells were lysed in a buffer containing0.1% SDS, 30 mM Tris, 150 mM NaCl, 10 mMEDTA, 0.5% sodium deoxycholate, 1% nonidet P-40,1 mM phenylmethylsulfonyl fluoride, 10 �g/mlaprotinin, 10 �g/ml leupeptin, and 1 mM Na3VO4,pH 7.5. The cell lysates were then centrifuged toremove insoluble materials. For immunoprecipitationexperiments, the same protein concentration of thesupernatants was used for immunoprecipitation withthe specified antibody for 2 h at 4°C. The entireprecipitates or the supernatants beforeimmunoprecipitation (20 �g protein) were separatedby SDS-PAGE and transferred to PVDF membranesusing a Bio-Rad Transblot apparatus. Themembranes were blocked in a buffer containing 50mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5%BSA, pH 7.5 for 30 min at 20°C. The membraneswere then probed with specified antibodies for 2 h at20°C. After washing the membranes in a buffercontaining 50 mM Tris, 150 mM NaCl, and 0.1%Tween 20, pH 7.5, blots were incubated withhorseradish peroxidase-linked second antibodyfollowed by enhanced chemiluminescence detectionusing ECL reagents, according to the manufacturer’sinstructions (Amersham Pharmacia Biotech Corp.).

2.4. DNA synthesis assay

Serum-starved A10 cells grown in 96 multi-well platewere incubated with various concentrations of PDGF.Then, the cells were treated with 10 mM 5-bromo-2�-de-oxyuridine (BrdU) for 3 h. BrdU incorporation intoDNA was measured by utilizing colorimetric reactionwith peroxidase-linked anti-BrdU antibody using CellProliferation ELISA kit according to the manufactur-er’s instructions (Boehringer Mannheim, Germany). Be-cause the peak of DNA synthesis in A10 cells wasobserved when BrdU was present for 3 h starting 20 hafter the addition of PDGF, BrdU incorporation wasassayed after the cells were incubated with PDGF for20 h.

2.5. PI3-kinase acti�ity assay

Serum-starved A10 cells grown in 10 cm dishes werestimulated with 167 pM PDGF at 37°C for the indi-cated times. The cells were solubilized in a buffercontaining 20 mM Tris, 137 mM NaCl, 1 mM MgCl2,1 mM CaCl2, 100 �M Na3VO4, 1% Nonidet-P40, 10%glycerol, 2 mM PMSF, and 100 �g/ml aprotinin, pH7.6. The cell lysates were then centrifuged at 10 000×gfor 20 min at 4°C to remove insoluble materials. Thesupernatants were immunoprecipitated with anti-PDGF�-receptor antibody for 2 h and then incubated with

Protein A sepharose for 1 h at 4°C. The immunoprecip-itates were washed twice with each of the followingbuffers: (i) PBS containing 1% Nonidet P-40, 100 �MNa3VO4, 1 mM dithiothreitol (DTT), pH 7.6; (ii) 100mM Tris, 500 mM LiCl, 100 �M Na3VO4, 1 mM DTT,pH 7.6; and (iii) 10 mM Tris, 100 mM NaCl, 1 mMEDTA, 1 mM DTT, pH 7.6. The phosphorylationreaction was started by adding 20 �l phosphatidylinosi-tol (PI) solution containing 0.5 mg/ml PI, 50 mMHEPES, 1 mM NaH2PO4, 1 mM EGTA, pH 7.6 fol-lowed by the addition of 10 �l of the reaction mixturecontaining 250 �M [�-32P]ATP (0.37 MBq/tube), 100mM HEPES, 50 mM MgCl2, pH 7.6 for 5 min at 22°C.The reaction was terminated by the addition of 15 �l 8M HCl. The PI products were extracted by adding 130�l of chloroform/methanol (1:1). After centrifugation ofthe sample, the organic phase was removed and spottedon Silica Gel 60 plate. The plates were developed inCHCl3: CH3OH: H2O: NH4OH (120:94: 22.6:4) anddried. The phosphorylated inositol was visualized byautoradiography, and 32P-phosphate incorporated intoinositol was determined by the Bio-Image Analyzer(Fuji Film, Tokyo, Japan).

2.6. Statistical analysis

The data are expressed as the means�S.E. P valueswere determined by Mann–Whitney method, and P�0.05 was considered statistically significant.

3. Results

3.1. Effect of high glucose and azaserine onPDGF-induced DNA synthesis

PDGF stimulated BrdU incorporation in A10 cells(Figs. 1 and 2). Incubation of A10 cells with 20 mMglucose for 1 or 2 week(s) did not apparently affect 167pM PDGF-induced BrdU incorporation. On the otherhand, further incubation of the cells in the continualpresence of 20 mM glucose including passage for 4weeks led to increased PDGF-induced BrdU incorpora-tion by 33�4% (P�0.05 vs. control). These increasesdid not originate from osmolarity induced by highglucose treatment, because addition of 14.5 mM manni-tol plus 5.5 mM glucose instead of 20 mM glucose didnot affect PDGF-induced BrdU incorporation. Since along period of high glucose treatment was required forthe enhancement of PDGF-induced BrdU incorpora-tion, glucose metabolites were considered as possiblecandidates in eliciting the enhancement. Azaserine isknown to inhibit the enzymatic activity of GFAT,which is the rate-limiting enzyme of the hexosaminebiosynthetic pathway. The effect of azaserine on highglucose enhancement of PDGF action was examined.


Fig. 1. Effect of high glucose and azaserine on PDGF-induced DNAsynthesis. A10 cells were cultured with 5.5 mM glucose (Control), 20mM glucose (HG), 5.5 mM glucose plus 14.5 mM mannitol (Manni-tol), or 20 mM glucose plus 500 nM azaserine for the indicatedweeks. 167 pM PDGF-stimulated BrdU incorporation in A10 cellswas assayed as described under Section 2. Results are the mean�S.E. of three separate experiments. * P�0.05 versus control group.

3.2. Effect of glucosamine on PDGF-induced DNAsynthesis

Since treatment with high glucose for 4 weeks wasrequired for the enhancement of PDGF action andazaserine inhibited its effects, it is possible thathexosamine biosynthesis is involved in the effects ofhigh glucose. Therefore, the effect of glucosaminetreatment on PDGF-induced DNA synthesis was ex-amined. PDGF stimulated BrdU incorporation in adose-dependent fashion with an ED50 value of 128�5 pM in A10 cells cultured in control medium. Incu-bation of the cells in culture media with glucosamineenhanced PDGF-stimulated BrdU incorporation in atime- and dose-dependent fashion. Treatment for 1week with 4 mM glucosamine significantly increased167 and 333 pM PDGF-induced BrdU incorporationby 46�6% (P�0.05) and 27�4% (P�0.05), respec-tively, compared with those in control culture. In ad-dition, PDGF sensitivity was also significantlyincreased with a leftward shift of the dose-responsecurve (ED50 value, 73�7 pM; P�0.05 vs. control)in the presence of 4 mM glucosamine (Fig. 2). Thesealterations did not originate from osmolarity inducedby glucosamine treatment, because addition of 4 mMmannitol instead of glucosamine again did not affectPDGF-stimulated BrdU incorporation (data notshown). These findings indicate that exposure to glu-cosamine itself enhances PDGF-stimulated DNA syn-thesis in A10 cells.

3.3. Effect of glucosamine on protein concentration andtyrosine phosphorylation of PDGF �-receptors

To clarify the mechanisms of glucosamine effect onincreased PDGF-stimulated BrdU incorporation, weexamined the protein concentration of PDGF �-recep-tors following glucosamine treatment. The number ofPDGF �-receptors in cell lysates was slightly de-creased by glucosamine treatment (Fig. 3A). Densito-metric analysis revealed that the density of PDGF�-receptors was decreased by 7–9%, although not sig-nificant, in the cells treated with glucosamine com-pared with control cells (Fig. 3C). Despite the slightlydecreased amount of PDGF �-receptors, tyrosinephosphorylation of the PDGF �-receptor was appar-ently increased by glucosamine treatment (Fig. 3B).PDGF-induced tyrosine phosphorylation of thePDGF �-receptor was increased by 22�2% (P�0.01) at 10 min and 20�5% (P�0.01) at 60 minafter PDGF stimulation in A10 cells cultured with 4mM glucosamine compared with those in control me-dia (Fig. 3D). These findings indicate that glu-cosamine enhances DNA synthesis by increasingtyrosine phosphorylation of the PDGF �-receptor.

Interestingly, co-incubation with azaserine inhibitedthe effect of high glucose on PDGF-induced BrdUincorporation (Fig. 1).

Fig. 2. Effect of glucosamine on PDGF-induced DNA synthesis. A10cells were cultured without (�) or with 1 mM (�), 2 mM (�), and4 mM (�) glucosamine for 1 week. BrdU incorporation in A10 cellswas assayed as described under Section 2. Dose–response curves forPDGF stimulation of BrdU incorporation are shown. Results are themean�S.E. of four separate experiments. * P�0.05 versus withoutglucosamine treatment group.


Fig. 3. Effect of glucosamine on protein concentration and tyrosine phosphorylation of PDGF �-receptors. A10 cells were cultured without (C)or with 4 mM glucosamine (G) for 1 week. The cells were serum-starved and treated with 167 pM PDGF at 37oC for the indicated times. (A)The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-PDGF �-receptor antibody. (B) The cell lysates wereimmunoprecipitated with anti-PDGF �-receptor antibody. The precipitates were subjected to SDS-PAGE and analyzed by immunoblotting withanti-phosphotyrosine antibody. The protein concentration of PDGF �-receptors (C) and tyrosine-phosphorylated PDGF �-receptors (D) werequantitated by densitometry. Results are the mean�S.E. of three separate experiments. c P�0.01 versus without glucosamine treatment group.

3.4. Effect of glucosamine on tyrosine phosphorylationof Shc and subsequent association with Grb2

The activated PDGF �-receptor phosphorylates ty-rosine residues of Shc, and the phosphorylated Shcassociates with Grb2 [11,12]. The Shc·Grb2 pathway hasbeen shown to be important in Ras-MAP kinase activa-tion leading to mitogenesis [12]. Since glucosamineenhanced tyrosine phosphorylation of the PDGF �-re-ceptor, the effect of glucosamine on PDGF-inducedtyrosine phosphorylation of Shc and Shc·Grb2 associa-tion was examined. In contrast to the findings of en-hanced tyrosine phosphorylation of the PDGF�-receptor, PDGF-induced Shc phosphorylation wasdecreased by treatment with glucosamine (Fig. 4A). Asshown in Fig. 4C, glucosamine treatment decreasedtyrosine phosphorylation of Shc by 34�5% (P�0.01)at 10 min and 39�10% (P�0.01) at 60 min after PDGFstimulation compared with those in control cells. Inaccordance with the decreased tyrosine phosphorylationof Shc, PDGF-induced Shc association with Grb2 wasreduced by glucosamine treatment (Fig. 4B). The findingsof densitometric analysis showed that Shc·Grb2 associa-tion was decreased by 17�4% (P�0.01) at 10 min and20�3% (P�0.01) at 60 min after PDGF stimulation inthe cells cultured with glucosamine compared with con-trol cells (Fig. 4D).

3.5. Effect of glucosamine on MAP kinase acti�ation

The Shc·Grb2 pathway has been shown to be impor-tant in Ras-MAP kinase activation [12]. We next exam-ined the effect of glucosamine on PDGF-induced MAPkinase activation. Activated p44-and p42-MAP kinasewere detected by immunoblotting the cell lysates withphospho-specific p42/44 MAP kinase antibody. PDGFinduced activation of both p44 and p42 MAP kinase inA10 cells. In accordance with the decreased tyrosinephosphorylation of Shc and subsequent association withGrb2, the amount of phosphorylated p44 and p42 MAPkinase was decreased in the cells cultured with glu-cosamine (Fig. 5A). It is notable that glucosaminetreatment did not apparently affect the protein concen-tration of p44 and p42 MAP kinase (data not shown).These findings are summarized in Fig. 5B and Fig. 5C.The amounts of phosphorylated p44 and p42 MAPkinase were decreased by 18�2% (P�0.01) and 8�2%(P�0.05) at 10 min, and 21�1% (P�0.01) and 20�5% (P�0.01) at 60 min, respectively, after PDGFstimulation in the cells cultured with glucosaminecompared with control cells. These findings indicatethat glucosamine does not increase PDGF-inducedmitogenesis via the Shc·Grb2 and MAP kinase path-ways.


3.6. Effect of glucosamine on PDGF �-receptorassociation with PLC�

PLC� is known to associate with PDGF �-receptorand is important for PDGF-induced DNA synthesis[8,10]. Therefore, we examined the effect of glu-cosamine on PDGF-induced PDGF �-receptor associa-tion with PLC�. PDGF �-receptor association withPLC� was seen in the basal state. Glucosamine treat-ment did not apparently affect the basal PDGF �-re-ceptor association with PLC� (Fig. 6A). PDGFstimulation increased PDGF �-receptor associationwith PLC� in a time-dependent manner. However, theassociation was not significantly affected by the glu-cosamine treatment as shown in Fig. 6A and Fig. 6B.These findings suggest that glucosamine-induced en-hancement of PDGF-induced BrdU incorporation isnot mediated via the PLC� pathway.

3.7. Effect of glucosamine on protein concentration ofp85 subunit of PI3-kinase and its association withPDGF �-receptor

PI3 kinase is an important downstream mediator ofthe PDGF �-receptor [8,9], and increasing evidenceindicates the importance of PI3-kinase in PDGF-in-duced mitogenesis in VSMCs [14–16]. Therefore, weexamined the effect of glucosamine on the proteinconcentration of p85 subunit of PI3-kinase. Glu-

cosamine treatment did not alter the protein concentra-tion of p85 regulatory subunit of PI3-kinase in thebasal and PDGF-stimulated states (Fig. 7A and Fig.7C). Since glucosamine-augmented tyrosine phosphory-lation of the PDGF �-receptor enhanced the activationof downstream molecules, we studied the effect ofglucosamine treatment on PDGF �-receptor associationwith p85 subunit. PDGF-stimulated PDGF �-receptorassociation with p85 subunit was enhanced by glu-cosamine treatment as shown in Fig. 7B. PDGF �-re-ceptor association with p85 subunit at 10 and 60 minfollowing PDGF stimulation was increased in the cellscultured with 4 mM glucosamine by 20�1% (P�0.01)and 14�3% (P�0.01), respectively, compared withthose in control cells (Fig. 7D).

3.8. Effect of glucosamine on PI3-kinase acti�ity

Since glucosamine increased PDGF �-receptor asso-ciation with the p85 subunit, the effect of glucosaminetreatment on PI3-kinase activity was examined. PI3-ki-nase activity was barely detected in the basal state, andPDGF stimulated PI3-kinase activity in the controlcells. In accordance with the increased PDGF �-recep-tor association with p85 subunit, PDGF-stimulatedPI3-kinase activity was enhanced by glucosamine treat-ment (Fig. 8A). Glucosamine increased PI3-kinase ac-tivity by 36�8% (P�0.01) at 10 min and 43�6%(P�0.01) at 60 min after PDGF stimulation compared

Fig. 4. Effect of glucosamine on PDGF-induced tyrosine phosphorylation of Shc and Shc·Grb2 association. A10 cells were cultured without (C)or with 4 mM glucosamine (G) for 1 week. The cells were serum-starved and treated with 167 pM PDGF at 37°C for the indicated times. Thecell lysates were immunoprecipitated with anti-Shc antibody. The precipitates were subjected to SDS-PAGE and analyzed by immunoblotting withanti-phosphotyrosine antibody (A) or anti-Grb2 antibody (B). The amount of tyrosine-phosphorylated Shc (C) and Grb2 associated with Shc (D)was quantitated by densitometry. Results are the mean�S.E. of three separate experiments. c P�0.01 versus without glucosamine treatmentgroup.


Fig. 5. Effect of glucosamine on PDGF-induced MAP kinase activity.A10 cells were cultured without (C) or with 4 mM glucosamine (G)for 1 week. The cells were serum-starved and treated with 167 pMPDGF at 37°C for the indicated times. The cell lysates were subjectedto SDS-PAGE and analyzed by immunoblotting with anti-phosphospecific MAP kinase antibody (A). Phosphorylated p44 and p42 MAPkinases are shown by arrows. The amount of phosphorylated p44 (B)and p42 (C) MAP kinases was quantitated by densitometry. Resultsare the mean�SE of three separate experiments. * P�0.05; c P�0.01 versus without glucosamine treatment group.

ities in A10 cells were almost completely inhibited bythe indicated concentrations of their respective in-hibitors (data not shown). Treatment of the cells withPLC inhibitor, U73122, showed a minimum effect onglucosamine-induced enhancement of PDGF-stimu-lated BrdU incorporation. MEK1 inhibitor, PD98059,had only slight inhibitory effects on BrdU incorpora-tion. In contrast, treatment with PI3-kinase inhibitor,LY294002, significantly inhibited glucosamine-en-hanced BrdU incorporation. At 833 pM of PDGFconcentration, the amount of BrdU incorporation wasinhibited by 56�3% by treatment with LY294002 (Fig.9A). The effects of pharmacological inhibitors onPDGF-induced BrdU incorporation are summarized inFig. 9B. PD98059 and LY294002 inhibited 167 pMPDGF-induced BrdU incorporation by 47�5% (P�

Fig. 6. Effect of glucosamine on PDGF-induced PDGF �-receptorassociation with PLC�. A10 cells were cultured without (C) or with 4mM glucosamine (G) for 1 week. The cells were serum-starved andtreated with 167 pM PDGF at 37°C for the indicated times. The celllysates were immunoprecipitated with anti-PDGF �-receptor anti-body. The precipitates were subjected to SDS-PAGE and analyzed byimmunoblotting with anti-PLC� antibody (A). The amount of PLC�associated with PDGF �-receptor (B) was quantitated by densitome-try. Results are the mean�S.E. of three separate experiments.

with control cells (Fig. 8B). These findings indicate theimportance of PI3-kinase in glucosamine-enhancedPDGF signaling.

3.9. Effect of pharmacological inhibitors onglucosamine-induced enhancement of PDGF-stimulatedBrdU incorporation

Intracellular molecules responsible for the enhance-ment of PDGF-induced DNA synthesis by glucosaminewere further examined using pharmacological inhibitorsof MEK1, PI3-kinase, and PLC. Their enzymatic activ-


Fig. 7. Effect of glucosamine on protein concentration of p85 subunit of PI3-kinase and PDGF-induced PDGF �-receptor association with p85.A10 cells were cultured without (C) or with 4 mM glucosamine (G) for 1 week. The cells were serum-starved and treated with 167 pM PDGFat 37°C for the indicated times. (A) The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-p85 regulatorysubunit of PI3-kinase antibody. (B) The cell lysates were immunoprecipitated with anti-PDGF �-receptor antibody. The precipitates weresubjected to SDS-PAGE and analyzed by immunoblotting with anti-p85 subunit antibody. The protein concentration of p85 (C) and the amountof p85 subunit associated with PDGF �-receptor (D) were quantitated by densitometry. Results are the mean�S.E. of three separate experiments.c P�0.01 versus without glucosamine treatment group.

0.01) and 54�3% (P�0.01), respectively, in the con-trol media. Thus, the inhibitory effects of PD98059 andLY294002 were comparable in the absence of glu-cosamine. U73122 also inhibited 167 pM PDGF-in-duced BrdU incorporation in the absence ofglucosamine, although the inhibitory effect was smallerthan PD98059 and LY294002. Importantly, LY294002markedly inhibited PDGF-induced BrdU incorporationalso in the presence of glucosamine, whereas PD98059and U73122 had only slight effects on the BrdU incor-poration in the presence of glucosamine compared withthose in its absence. These findings indicate that al-though MAP kinase, PLC�, and PI3-kinase are allinvolved in PDGF-induced DNA synthesis under con-trol culture conditions, PI3-kinase plays a more impor-tant role in glucosamine enhancement of PDGFsignaling.

4. Discussion

Since proliferation of VSMCs plays a key role in thedevelopment of atherosclerosis, the effect of high glu-cose on cultured VSMC growth has been studied [31–35]. Previous reports indicated that high glucosetreatment increased proliferation of primary and sub-cultured VSMCs [31–35]. Cell cycle analysis suggested

the role of glucose as a progression factor, but not as acompetent factor in the proliferation of VSMCs [31,34].Although the mechanisms by which high glucose treat-ment causes growth promotion in VSMCs are uncer-tain, several possibilities have been proposed. Firstly,protein kinase C (PKC) appears to be involved in thiseffect, because high glucose-enhanced expression ofPDGF-� receptors and proliferation of VSMCs weresuppressed by treatment with PKC inhibitor [31,35].Secondly, involvement of the polyol pathway is sug-gested by the fact that an aldose reductase inhibitorprevented growth promotion of VSMCs induced byhigh glucose treatment [33,34]. Thirdly, the oxidativestress due to increased intracellular NADH/NAD+ byhigh glucose treatment appears to cause the promotionof cell growth [34]. In addition to inducing these abnor-malities, one can speculate that glucose metabolites viathe hexosamine biosynthetic pathway may also be in-volved in growth promotion [17]. In this regard, glu-cosamine, rather than glucose, is reported to be apotent transcriptional stimulator of TGF-� gene, andglucose metabolism to glucosamine appears to be re-quired for the stimulation in VSMCs [27–29]. Theseprevious findings prompted us to examine the effect ofglucosamine on PDGF-induced DNA synthesis, whichplays a crucial role in the development of atherosclero-sis [2–6]. Importantly, our findings clearly demon-


strated that glucosamine treatment for 1 week en-hanced PDGF-induced DNA synthesis in A10 cells.This indicates that the hexosamine biosynthetic path-way appears not only to be involved in promotinginsulin resistance [18–26], but also plays an importantrole in the promotion of VSMC proliferation.

The glucosamine concentration used in this studywas chosen to mimic metabolic disturbances observedin previous studies with intravenous in vivo glu-cosamine infusion into rats [22,36,37]. Althoughserum glucosamine concentrations in intact animalsare generally below detectable levels (�0.05 mM)[22,36,37], the addition of glucosamine increased theamount of intracellular UDP-N-acetylhexosaminecomposed of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, which was observed by chro-matography and 31P-labeled nuclear magneticresonance [38,39]. In addition, the predominantmetabolite that increases in skeletal muscle followingin vivo glucosamine infusion into rat is known to beUDP-N-acetylglucosamine [36]. UDP-N-acetylglu-cosamine is known to be an obligatory intermediate

Fig. 9. Effect of pharmacological inhibitors on glucosamine-inducedenhancement of PDGF-stimulated BrdU incorporation. (A) A10 cellscultured with 4 mM glucosamine for 1 week were serum-starved andtreated without (�) or with either, 50 �M MEK1 inhibitor PD98059(�), 10 �M PI3-kinase inhibitor LY294002 (�), or 500 nM PLCinhibitor U73122 (�). BrdU incorporation in A 10 cells was assayedas described under Section 2. Dose–response curves for PDGFstimulation of BrdU incorporation are shown. Results are themean�S.E. of three separate experiments. (B) A10 cells culturedwithout (C) or with 4 mM glucosamine (G) for 1 week were serum-starved and treated with either, 50 �M PD98059, 10 �M LY294002,or 500 nM U73122. The inhibitory effect of these inhibitors on 167pM PDGF-induced BrdU incorporation is shown. Results are ex-pressed as percent inhibition of BrdU incorporation in the presenceof various inhibitors, and are the mean�S.E. of three separateexperiments.

Fig. 8. Effect of glucosamine on PDGF-induced PI3-kinase activity.A10 cells were cultured without (C) or with 4 mM glucosamine (G)for 1 week. The cells were serum-starved and treated with 167 pMPDGF at 37°C for the indicated times. PI3-kinase activity in theanti-PDGF �-receptor immunoprecipitates was measured. (A) A rep-resentative autoradiogram of the thin-layer chromatography isshown. The origin and the migration of labeled PtdIns(3)P areindicated by arrows. (B) The amount of labeled lipid products on theautoradiograms was quantitated by Bio-Image Analyzer. Results areshown as mean�S.E. of three experiments. cP�0.01 versus with-out glucosamine treatment group.

in the glycosylation and important for processing ofglycoproteins in the endoplasmic reticulum and Golgisystem [38]. Taken together, UDP-N-acetylglu-cosamine may be the glucosamine metabolite respon-sible for exhibiting the enhanced PDGF-inducedDNA synthesis observed in the current studies.


The enhancement of PDGF-induced DNA synthesiswas due to augmentation of PDGF �-receptor phos-phorylation and subsequent alterations in signal trans-duction leading to DNA synthesis. These findings maynot be in agreement with the previous finding that ahigh concentration of glucose enhanced PDGF signal-ing at the level of PDGF-� receptor expression inrabbit VSMCs, while the tyrosine phosphorylation ofPDGF- � receptor was not studied [35]. Because theincreased expression of PDGF-� receptor was also ob-served in A10 cells by high glucose treatment for 4weeks (unpublished data), but not with glucosamine for1 week, enhancement of PDGF signaling at the level ofPDGF-� receptor phosphorylation may be specific tothe effect of glucosamine. Therefore, glucosamine ap-pears to contribute to the development of atherosclero-sis by mechanisms that differ, at least in part, fromthose already known for high glucose-induced growthpromotion of VSMC. In addition, glucosamine mayalso be similarly involved in progression of atheroscle-rosis without adult onset of diabetes as well as indiabetes.

It is known that multiple signaling pathways initiatedfrom the activated PDGF �-receptor exist in DNAsynthesis. Of these pathways, p21ras plays a pivotalrole in PDGF-induced DNA synthesis [40,41]. Al-though tyrosine-phosphorylated PDGF �-receptor andShc may both independently bind to Grb2, which ispresent as a preformed complex with guanine nucle-otide exchange factor Sos for p21Ras activation, Shc isshown to be a predominant coupling molecule for Grb2in vascular smooth muscle A10 cells [12]. Thus, tyrosinephosphorylated PDGF �-receptor augmentation ofPDGF signaling via Shc phosphorylation is a possiblemechanism of glucosamine enhancement of PDGF-in-duced DNA synthesis. However, this was not the casein our study. Despite the augmented tyrosine phospho-rylation of the PDGF �-receptor, the amount of ty-rosine phosphorylation of Shc and subsequentassociation of Shc with Grb2 were unexpectedly de-creased as shown in Fig. 4. PDGF-induced MAP ki-nase activation was also decreased by glucosaminetreatment in A10 cells as shown in Fig. 5. Thesefindings indicate that the effect of glucosamine onPDGF-induced DNA synthesis is not mediated via theShc·Grb2 pathway and MAP kinase. This notion isfurther confirmed by the fact that blockade of MAPkinase cascade by MEK1 inhibitor PD98059 onlypartly inhibited the effect of glucosamine. It has beenreported that PLC� is involved in PDGF-induced DNAsynthesis in VSMCs [13,14]. Microinjection of eitherGST fusion proteins containing SH2 domain of PLC�or anti-PLC� specific antibody inhibited PDGF-in-duced DNA synthesis [10]. However, our findingsdemonstrated that glucosamine treatment did not affectPDGF-induced PDGF �-receptor association with

PLC�. In addition, treatment of A10 cells with PLCinhibitor did not significantly alter the glucosamineeffect. Therefore, the PLC� mediated signaling pathwaydoes not appear to be involved in glucosamine enhance-ment of PDGF-induced DNA synthesis in A10 cells.On the other hand, PI3-kinase is also known to beimportant in PDGF-induced DNA synthesis [8,9,14–16], and p70 S6-kinase, a downstream mediator ofPI3-kinase, is crucial for S phase entry [42]. In contrastto the findings of Shc·Grb2 and PLC� mediated path-ways, glucosamine enhanced PDGF-induced PDGF �-receptor association with the p85 regulatory subunit ofPI3-kinase and PI3-kinase activation. In addition, aspecific inhibitor of PI3-kinase, LY294002, clearly in-hibited PDGF-induced DNA synthesis augmented byglucosamine. Taken together, PI3-kinase appears to bea crucial mediator for glucosamine enhancement ofPDGF-induced DNA synthesis in vascular muscle A10cells. These findings support a recent in vivo findingthat association of PI3-kinase with phosphorylatedPDGF �-receptors was increased in neointima in theballoon-injured rat carotid artery [43].

Although the mechanisms by which increased ty-rosine phosphorylation of the PDGF �-receptor onlyactivates the PI3-kinase pathway are uncertain, it isinteresting to hypothesize a glycosylated modificationof the intracellular domain of PDGF �-receptor andp85 subunit of PI3-kinase induced by glucosaminetreatment. Because apparent glycosylation of PDGF�-receptor and p85 subunit of PI3-kinase, but not ofShc and PLC�, was found following glucosamine treat-ment (unpublished data), glycosylation may change thetertiary structures leading to specifically altered stoi-chiometry of the PDGF �-receptor binding to p85subunit. Although our findings demonstrated the im-portance of PI3-kinase in the effect of glucosamine, wecan not rule out the possible roles of other adaptormolecules including Src, SHP2, and Nck, which canassociate with the activated PDGF �-receptor. The roleof these binding molecules to the activated PDGF�-receptor remains to be investigated to further clarifythe mechanism of glucosamine enhancement of PDGFsignaling.

In summary, these findings suggest the involvementof glucosamine, an intracellular metabolite of glucose,in the development of atherosclerosis through enhancedPDGF signaling. Glucosamine appears to enhancePDGF-induced DNA synthesis specifically via the PI3-kinase pathway in vascular smooth muscle A10 cells.

Acknowledgements

This work was supported in part by the Grant-in-Aidfor Scientific Research (C) from Japan Society for thePromotion of Science and Japan Research Foundationfor Clinical Pharmacology (to T.S.).


References

[1] American Diabetes Association, 1989. Role of cardiovascularrisk factors in prevention and treatment of macrovascular dis-ease in diabetes. Diabetes Care, 12, 573-579.

[2] Ross R. The pathogenesis of atherosclerosis: a perspective forthe 1990s. Nature 1993;362:801–9.

[3] Ross R, Masuda J, Raines EW, Gown AM, Katsuda S, Sasa-hara M, Malden LT, Masuko H, Sato H. Localization ofPDGF-B protein in macrophages in all phases of atherogenesis.Science 1990;248:1009–12.

[4] Ferns GAA, Raines EW, Sprugel KH, Motani AS, Reidy MA,Ross R. Inhibition of neointimal smooth muscle accumulationafter angioplasty by an antibody to PDGF. Science1991;253:1129–32.

[5] Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, ClowesAW. Platelet-derived growth factor promotes smooth musclemigration and intimal thickening in a rat model of balloonangioplasty. J Clin Invest 1992;89:507–11.

[6] Sirois MG, Simons M, Edelman ER. Antisense oligonucleotideinhibition of PDGFR-� receptor subunit expression directs sup-pression of intimal thickening. Circulation 1997;95:669–76.

[7] Claesson-Welsh L. Platelet-derived growth factor receptor sig-nals. J Biol Chem 1994;269:32023–6.

[8] Valius M, Kazlauskas A. Phospholipase C-�1 and phosphatidyli-nositol 3 kinase are the downstream mediators of the PDGFreceptor’s mitogenic signal. Cell 1993;73:321–34.

[9] Roche S, Koegl M, Courtneidge SA. The phosphatidylinositol3-kinase � is required for DNA synthesis induced by some, butnot all, growth factors. Proc Natl Acad Sci USA 1994;91:9185–9.

[10] Roche S, McGlade J, Jones M, Gish GD, Pawson T, Court-neidge SA. Requirement of phospholipase C�, the tyrosine phos-phatase Syp and the adaptor proteins Shc and Nck forPDGF-induced DNA synthesis: evidence for the existence ofRas-dependent and Ras-independent pathways. EMBO J1996;15:4940–8.

[11] Yokote K, Mori S, Hansen K, McGlade J, Pawson T, HeldinC-H, Claesson-Welsh L. Direct interaction between Shc and theplatelet-derived growth factor �-receptor. J Biol Chem1994;269:15337–43.

[12] Benjamin CW, Jones DA. Platelet-derived growth factor stimu-lates growth factor receptor binding protein-2 association withShc in vascular smooth muscle cells. J Biol Chem1994;269:30911–6.

[13] Homma Y, Sakamoto H, Tsunoda M, Aoki M, Takenawa T,Ooyama T. Evidence for involvement of phospholipase C-�2 insignal transduction of platelet-derived growth factor in vascularsmooth-muscle cells. Biochem J 1993;290:649–53.

[14] Ali S, Dorn GW. Patterns of tyrosine phosphorylation differ invascular hypertrophy and hyperplasia. Am J Physiol1994;267:C1674–81.

[15] Bacqueville D, Casagrande F, Perret B, Chap H, Darbon J-M,Breton-Douillon M. Phosphatidylinositol 3-kinase inhibitorsblock aortic smooth muscle cell proliferation in mid-late G1phase: effect on cyclin-dependent kinase 2 and the inhibitoryprotein p27KIP1. Biochem Biophys Res Commun 1998;244:630–6.

[16] Thyberg J. Tyrphostin A9 and wortmannin perturb the Golgicomplex and block proliferation of vascular smooth muscle cells.Eur J Cell Biol 1998;76:33–42.

[17] McClain DA, Crook ED. Hexosamines and insulin resistance.Diabetes 1996;45:1003–9.

[18] Marshall S, Bacote V, Traxinger RR. Discovery of a metabolicpathway mediating glucose-induced desensitization of the glu-cose transport system: role of hexosamine biosynthesis in theinduction of insulin resistance. J Biol Chem 1991;266:4706–12.

[19] Crook ED, Daniels MC, Smith TM, McClain DA. Regulationof insulin-stimulated glycogen synthase activity by overexpres-sion of glutamine: fructose-6-phosphate amidotransferase in rat-1 fibroblasts. Diabetes 1993;42:1289–96.

[20] Robinson KA, Sens DA, Buse MG. Pre-exposure to glucosamineinduces insulin resistance of glucose transport and glycogensynthesis in isolated rat skeletal muscles: study of mechanisms inmuscle and Rat-1 fibroblasts overexpressing the human insulinreceptor. Diabetes 1993;42:1333–46.

[21] Crook ED, Zhou J, Daniels M, Neidigh JL, McClain DA.Regulation of glycogen synthase by glucose, glucosamine, andglutamine:fructose-6-phosphate amidotransferase. Diabetes1995;44:314–20.

[22] Rossetti L, Hawkins M, Chen W, Gindi J, Barzilai N. In vivoglucosamine infusion induces insulin resistance in normo-glycemic but not in hyperglycemic conscious rats. J Clin Invest1995;96:132–40.

[23] Baron AD, Zhu J-S, Zhu J-H, Weldon H, Maianu L, GarveyWT. Glucosamine induces insulin resistance in vivo by affectingGlut4 translocation in skeletal muscle: implications for glucosetoxicity. J Clin Invest 1995;96:2792–801.

[24] Crook ED, McClain DA. Regulation of glycogen synthase andprotein phosphatase-1 by hexosamines. Diabetes 1996;45:322–7.

[25] Hebert LF, Daniels MC, Zhou J, Crook ED, Turner RL,Simmons ST, Neidigh JL, Zhu J-S, Baron AD, McClain DA.Overexpression of glutamine:fructose-6-phosphate amidotrans-ferase in transgenic mice leads to insulin resistance. J Clin Invest1996;98:930–6.

[26] Hawkins M, Angelov I, Liu R, Barzilai N, Rossetti L. The tissueconcentration of UDP-N-acetylglucosamine modulates the stim-ulatory effect of insulin on skeletal muscle glucose uptake. J BiolChem 1997;272:4889–95.

[27] McClain DA, Paterson AJ, Roos MD, Wei X, Kudlow JE.Glucose and glucosamine regulate growth factor gene expressionin vascular smooth muscle cells. Proc Natl Acad Sci USA1992;89:8150–4.

[28] Daniels MC, Kansal P, Smith TM, Paterson AJ, Kudlow JE,McClain DA. Glucose regulation of transforming growth factor-� expression is mediated by products of the hexosamine biosyn-thesis pathway. Mol Endocrinol 1993;7:1041–8.

[29] Sayeski PP, Kudlow JE. Glucose metabolism to glucosamine isnecessary for glucose stimulation of transforming growth factor-� gene transcription. J Biol Chem 1996;271:15237–43.

[30] Kolm-Litty V, Sauer U, Nerlich A, Lehmann R, Schleicher ED.High glucose-induced transforming growth factor �1 productionis mediated by the hexosamine pathway in porcine glomerularmesangial cells. J Clin Invest 1998;101:160–9.

[31] Hiroishi G, Kobayashi S, Nishimura J, Inomata H, Kanaide H.High D-glucose stimulates the cell cycle from the G1 to the S andM phases, but has no competent effect on the G0 phase, invascular smooth muscle cells. Biochem Biophys Res Commun1995;211:619–26.

[32] Natarajan R, Gonzales N, Xu L, Nadler JL. Vascular smoothmuscle cells exhibit increased growth in response to elevatedglucose. Biochem Biophys Res Commun 1992;187:552–60.

[33] Graier WF, Grubenthal I, Dittrich P, Wascher TC, KostnerGM. Intracellular mechanism of D-glucose-induced modulationof vascular cell proliferation. Eur J Pharmacol 1995;294:221–9.

[34] Yasunari K, Kohno M, Kano H, Yokokawa K, Horio T,Yoshikawa J. Aldose reductase inhibitor prevents hyperprolifer-ation and hypertrophy of cultured rat vascular smooth musclecells induced by high glucose. Arterioscler Thromb Vasc Biol1995;15:2207–12.

[35] Inaba T, Ishibasi S, Gotoda T, Kawamura M, Morino N,Nojima Y, Kawakami M, Yazaki Y, Yamada N. Enhancedexpression of platelet-derived growth factor-� receptor by highglucose: involvement of platelet-derived growth factor in diabeticangiopathy. Diabetes 1996;45:507–12.


[36] Patti M-E, Virkamaki A, Landaker EJ, Kahn CR, Yki-JarvinenH. Activation of the hexosamine pathway by glucosamine in vivoinduces insulin resistance of early postreceptor insulin signalingevents in skeletal muscle. Diabetes 1999;48:1562–71.

[37] Hawkins M, Hu M, Yu J, Eder H, Vuguin P, She L, Barzilai N,Leiser M, Backer JM, Rossetti L. Discordant effects of glu-cosamine on insulin-stimulated glucose metabolism and phos-phatidylinositol 3-kinase activity. J Biol Chem 1999;274:31312–9.

[38] Wice BM, Trugnan G, Pinto M, Rousset M, Chevalier G,Dussaulx E, Lacroix B, Zweibaum A. The intracellular accumu-lation of UDP-N-acetylhexosamine is concomitant with the inabil-ity of human colon cancer cells to differentiate. J Biol Chem1985;260:139–46.

[39] Pederson NV, Knop RH, Miller WM. UDP-N-acetylhexosamine

modulation by glucosamine and uridine in NCI N-417 variantsmall cell lung cancer cells: 31P nuclear magnetic resonance results.Cancer Res 1992;52:3782–6.

[40] Satoh T, Endo M, Nakafuku M, Nakamura S, Kaziro Y.Platelet-derived growth factor stimulates formation of activep21ras·GTP complex in Swiss mouse 3T3 cells. Proc Natl AcadSci USA 1990;87:5993–7.

[41] Satoh T, Nakafuku M, Kaziro Y. Function of Ras as a molecularswitch in signal transduction. J Biol Chem 1992;267:24149–52.

[42] Lane HA, Fernandez A, Lamb NJC, Thomas G. p70s6k functionis essential for G1 progression. Nature 1993;363:170–2.

[43] Panek RL, Dahring TK, Olszewski BJ, Keiser JA. PDGF receptorprotein tyrosine kinase expression in the balloon-injured ratcarotid artery. Arterioscler Thromb Vasc Biol 1997;17:1283–8.

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Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells