Title: Glycosylation Mediates Up-regulation of a Potent Antiangiogenic and Proatherogenic Protein, Thrombospondin-1, by Glucose in Vascular Smooth Muscle Cells
Abstract: Accelerated development of atherosclerotic lesions remains the most frequent and dangerous complication of diabetes, accounting for 80% of deaths among diabetics. However, our understanding of the pathways mediating glucose-induced gene expression in vascular cells remains controversial and incomplete. We have identified an intracellular metabolic pathway activated by high glucose in human aortic smooth muscle cells that mediates up-regulation of thrombospondin-1 (TSP-1). TSP-1 is a potent antiangiogenic and proatherogenic protein that may represent an important link between diabetes and vascular complications. Using different glucose analogs and metabolites sharing distinct, limited metabolic steps with glucose, we demonstrated that activation of TSP-1 transcription is mediated by the hexosamine pathway of glucose catabolism, possibly resulting in modulation of the activity of nuclear proteins activity through their glycosylation. Specific inhibitors of glutamine: fructose 6-phosphate amidotransferase (GFAT), an enzyme controlling the hexosamine pathway, as well as direct inhibitors of protein glycosylation efficiently inhibited TSP-1 transcription and the activity of a TSP-1 promoter-reporter construct stimulated by high glucose. Overexpression of recombinant GFAT resulted in increased TSP-1 levels. Pharmacological inhibition of GFAT or protein glycosylation inhibited increased proliferation of human aortic smooth muscle cells caused by glucose. We have demonstrated that the hexosamine metabolic pathway mediates up-regulation of TSP-1 by high glucose. Our results suggest that the hexosamine pathway and intracellular glycosylation may control important steps in initiation and development of atherosclerotic lesions. Accelerated development of atherosclerotic lesions remains the most frequent and dangerous complication of diabetes, accounting for 80% of deaths among diabetics. However, our understanding of the pathways mediating glucose-induced gene expression in vascular cells remains controversial and incomplete. We have identified an intracellular metabolic pathway activated by high glucose in human aortic smooth muscle cells that mediates up-regulation of thrombospondin-1 (TSP-1). TSP-1 is a potent antiangiogenic and proatherogenic protein that may represent an important link between diabetes and vascular complications. Using different glucose analogs and metabolites sharing distinct, limited metabolic steps with glucose, we demonstrated that activation of TSP-1 transcription is mediated by the hexosamine pathway of glucose catabolism, possibly resulting in modulation of the activity of nuclear proteins activity through their glycosylation. Specific inhibitors of glutamine: fructose 6-phosphate amidotransferase (GFAT), an enzyme controlling the hexosamine pathway, as well as direct inhibitors of protein glycosylation efficiently inhibited TSP-1 transcription and the activity of a TSP-1 promoter-reporter construct stimulated by high glucose. Overexpression of recombinant GFAT resulted in increased TSP-1 levels. Pharmacological inhibition of GFAT or protein glycosylation inhibited increased proliferation of human aortic smooth muscle cells caused by glucose. We have demonstrated that the hexosamine metabolic pathway mediates up-regulation of TSP-1 by high glucose. Our results suggest that the hexosamine pathway and intracellular glycosylation may control important steps in initiation and development of atherosclerotic lesions. Thrombospondins are matricellular proteins that regulate cell-cell and cell-matrix interactions (1Bornstein P. J. Clin. Investig. 2001; 107: 929-934Crossref PubMed Scopus (409) Google Scholar, 2Adams J.C. Annu. Rev. Cell Dev. Biol. 2001; 17: 25-51Crossref PubMed Scopus (328) Google Scholar). Recent genetic association studies link the thrombospondin (TSP) 2The abbreviations used are: TSP, thrombospondin; THBS1, TSP-1 gene; SMC, smooth muscle cell(s); HASMC, human aortic SMC; GFAT, glutamine:fructose 6-phosphate amidotransferase; DON, 6-diazo 5-oxonorleucine; BG, benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside; STZ, streptozotocin; PUGNAc, O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate; XTT, sodium 3,3′-{1-[phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro); USF, upstream stimulatory factor; siRNA, small interfering RNA; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum. protein family to the development of atherosclerotic lesions (3Topol E.J. McCarthy J. Gabriel S. Moliterno D.J. Rogers W.J. Newby L.K. Freedman M. Metivier J. Cannata R. O'Donnell C.J. Kottke-Marchant K. Murugesan G. Plow E.F. Stenina O. Daley G.Q. Circulation. 2001; 104: 2641-2644Crossref PubMed Scopus (258) Google Scholar, 4Kato T.Y.A. Murase Y. Hirashiki A. Noda A. Yamada Y. Circulation. 2003; 108: IV-712Google Scholar, 5Walton B.W.J. Coresh J. Boerwinkle E. Circulation. 2003; 108: IV-771Google Scholar, 6Cui J. Randell E. Renouf J. Sun G. Han F.Y. Younghusband B. Xie Y.G. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 183-184Crossref PubMed Google Scholar, 7Yamada Y. Izawa H. Ichihara S. Takatsu F. Ishihara H. Hirayama H. Sone T. Tanaka M. Yokota M. N. Engl. J. Med. 2002; 347: 1916-1923Crossref PubMed Scopus (606) Google Scholar, 8Boekholdt S.M. Trip M.D. Peters R.J. Engelen M. Boer J.M. Feskens E.J. Zwinderman A.H. Kastelein J.J. Reitsma P.H. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 24-27Crossref PubMed Google Scholar, 9Wessel J. Topol E.J. Ji M. Meyer J. McCarthy J.J. Am. Heart J. 2004; 147: 905-909Crossref PubMed Scopus (51) Google Scholar, 10Zwicker J.I. Peyvandi F. Palla R. Lombardi R. Canciani M.T. Cairo A. Ardissino D. Bernardinelli L. Bauer K.A. Lawler J. Mannucci P. Blood. 2006; 108: 1280-1283Crossref PubMed Scopus (52) Google Scholar). TSP-1 was found in early atherosclerotic lesions (11Riessen R. Kearney M. Lawler J. Isner J.M. Am. Heart J. 1998; 135: 357-364Crossref PubMed Scopus (94) Google Scholar), in injured vascular walls (12Raugi G.J. Mullen J.S. Bark D.H. Okada T. Mayberg M.R. Am. J. Pathol. 1990; 137: 179-185PubMed Google Scholar, 13Sajid M. Hu Z. Guo H. Li H. Stouffer G.A. J. Investig. Med. 2001; 49: 398-406Crossref PubMed Scopus (27) Google Scholar), and in cardiac allografts where its expression correlated with the degree of vasculopathy (14Zhao X.M. Hu Y. Miller G.G. Mitchell R.N. Libby P. Circulation. 2001; 103: 525-531Crossref PubMed Scopus (51) Google Scholar). The genetic disruption of TSP-1 reduced the atherosclerotic lesion area in the mouse model of atherosclerosis and suggested an important role for TSP-1 in the evolution of plaque and its composition (15Cody M. Lawler J. O'Neal T. Agah R. Circulation. 2005; 112: II-164Google Scholar). In both in vivo and in vitro studies TSP-1 induced proliferation of vascular smooth muscle cells (SMC) (16Majack R.A. Cook S.C. Bornstein P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9050-9054Crossref PubMed Scopus (210) Google Scholar, 17Majack R.A. Goodman L.V. Dixit V.M. J. Cell Biol. 1988; 106: 415-422Crossref PubMed Scopus (190) Google Scholar), and both TSP-1 and TSP-2 inhibited growth of endothelial cells (18Lawler J. J. Cell. Mol. Med. 2002; 6: 1-12Crossref PubMed Scopus (458) Google Scholar, 19Guo N. Krutzsch H.C. Inman J.K. Roberts D.D. Cancer Res. 1997; 57: 1735-1742PubMed Google Scholar, 20Armstrong L.C. Bjorkblom B. Hankenson K.D. Siadak A.W. Stiles C.E. Bornstein P. Mol. Biol. Cell. 2002; 13: 1893-1905Crossref PubMed Scopus (91) Google Scholar, 21Isenberg J.S. Calzada M.J. Zhou L. Guo N. Lawler J. Wang X.Q. Frazier W.A. Roberts D.D. Matrix Biol. 2005; 24: 110-123Crossref PubMed Scopus (50) Google Scholar); both effects are considered proatherogenic. Previous studies have documented increased TSP-1 levels in the plasma and kidneys of diabetic patients and diabetic animal models (22Bayraktar M. Dundar S. Kirazli S. Teletar F. J. Int. Med. Res. 1994; 22: 90-94Crossref PubMed Scopus (33) Google Scholar, 23Tschoepe D. Rauch U. Schwippert B. Horm. Metab. Res. 1997; 29: 631-635Crossref PubMed Scopus (86) Google Scholar, 24Olson B.A. Day J.R. Laping N.J. Pharmacology. 1999; 58: 200-208Crossref PubMed Scopus (24) Google Scholar, 25Murphy M. Godson C. Cannon S. Kato S. Mackenzie H.S. Martin F. Brady H.R. J. Biol. Chem. 1999; 274: 5830-5834Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). In mesangial cells, the level of TSP-1 was upregulated by glucose by a transcriptional mechanism (26Wang S. Wu X. Lincoln T.M. Murphy-Ullrich J.E. Diabetes. 2003; 52: 2144-2150Crossref PubMed Scopus (53) Google Scholar, 27Wang S. Skorczewski J. Feng X. Mei L. Murphy-Ullrich J.E. J. Biol. Chem. 2004; 279: 34311-34322Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 28Poczatek M.H. Hugo C. Darley-Usmar V. Murphy-Ullrich J.E. Am. J. Pathol. 2000; 157: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). We have recently reported increased levels of TSP-1 in the blood vessels of diabetic animals (29Stenina O.I. Krukovets I. Wang K. Zhou Z. Forudi F. Penn M.S. Topol E.J. Plow E.F. Circulation. 2003; 107: 3209-3215Crossref PubMed Scopus (113) Google Scholar). Moreover, TSP-1 was up-regulated by high glucose in vitro in major cell types from large blood vessels. These observations suggest that TSP-1 represents an important link between diabetes, hyperglycemia, and accelerated atherogenesis. A large number of clinical studies and trials have conclusively identified hyperglycemia as an independent risk factor for development of both micro- and macrovascular complications (30Nathan D.M. Lachin J. Cleary P. Orchard T. Brillon D.J. Backlund J.Y. O'Leary D.H. Genuth S. N. Engl. J. Med. 2003; 348: 2294-2303Crossref PubMed Scopus (758) Google Scholar, 31Malmberg K. BMJ. 1997; 314: 1512-1515Crossref PubMed Scopus (1213) Google Scholar, 32Barzilay J.I. Spiekerman C.F. Kuller L.H. Burke G.L. Bittner V. Gottdiener J.S. Brancati F.L. Orchard T.J. O'Leary D.H. Savage P.J. Diabetes Care. 2001; 24: 1233-1239Crossref PubMed Scopus (80) Google Scholar, 33Ohkubo Y. Kishikawa H. Araki E. Miyata T. Isami S. Motoyoshi S. Kojima Y. Furuyoshi N. Shichiri M. Diabetes Res. Clin. Pract. 1995; 28: 103-117Abstract Full Text PDF PubMed Scopus (2863) Google Scholar). Recently, the Epidemiology of Diabetes Intervention and Complications study reported that, as compared with conventional therapy, intensive glycemic control reduced the risk of the most serious cardiovascular events such as heart attacks, stroke, and death by nearly 60%. These findings clearly underscore the importance of hyperglycemia as a critical player in the development of pathogenic complications associated with diabetes. Glucose regulates the expression of a number of vascular genes, and many of them have been linked to the development of atherosclerosis and abnormal angiogenesis (34Stenina O.I. Curr. Pharm. Des. 2005; 11: 2367-2381Crossref PubMed Scopus (38) Google Scholar). However, the molecular mechanisms activated by glucose that lead to changes in the gene expression profile in vascular cells remain controversial and incomplete. In this study we have undertaken an unbiased approach to identify specific molecular mechanisms that mediate the upregulation of TSP-1 expression in response to high glucose in cultured HASMC. Using different glucose analogs and metabolites sharing distinct, limited metabolic steps with glucose, we found that the hexosamine pathway of glucose breakdown controls the expression of TSP-1 in HASMC. Our results demonstrate that the transcriptional regulation of TSP-1 is mediated by protein glycosylation, which is increased as a result of activation of the hexosamine pathway. Stimulation of Cultured SMC with Glucose and Glucose Analogs—Primary HASMC isolates, kindly provided by Dr. Donald Jacobsen and Dr. Edward F. Plow (Cleveland Clinic, Cleveland, OH) were grown to confluence in DMEM/F-12 medium with 10% fetal bovine serum (FBS). Cells with passage numbers between 3 and 12 were used in all experiments. 24 h before initiation of experiments, media were changed to low glucose (5 mm) DMEM supplemented with 0.2% FBS. Cells were stimulated with 30 mm glucose, fructose, mannose, galactose, 2-deoxyglucose, dihydroxyacetone, l-glucose, 2 mm glucosamine, 5 mm streptozotocin (STZ), and 100 μm O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) for 24 h. mRNA Stability Assay—Control cells or glucose-stimulated cells were treated with 5 μg/ml actinomycin D and then lysed at different time points from 20 min to 2 h after initiation of actinomycin D treatment. mRNA levels at each time point were quantified by densitometric scans of Northern blots. Northern Blots—Total RNA was extracted using TriZol reagent (Invitrogen). Isolated RNA was stored in diethyl pyrocarbonate-treated water at -80 °C. The purity and concentration were determined by measuring the optical density at 260 and 280 nm before use. The optical density ratio at 260/280 ranged from 1.7 to 2.0. For Northern blot analysis, 10 μg of SMC RNA was electrophoresed in 16.7% agarose-formaldehyde gels, transferred to nylon membranes (PerkinElmer Life Sciences), and hybridized to appropriate 32P-labeled cDNA probes using a Random prime labeling kit (Amersham Biosciences). The cDNA probe was a 600-bp fragment of TSP-1 cDNA corresponding to the N-terminal part of TSP-1 protein that has no homology with other thrombospondins. Membranes were prehybridized in ExpressHyb Hybridization solution (BD Biosciences) for 1 h at 68 °C. The heat-denatured cDNA probe was added, and hybridization was performed at 68 °C for 2 h. The membranes were then washed and exposed to Kodak BioMax MR film at -80 °C. Signal intensity was quantified using a Digital Science Imaging System (Version 2.0.1, Eastman Kodak Co.). Nuclear Run-on Assay—Nuclear run-on transcriptional assays were performed as described by Greenberg and Ziff (35Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-438Crossref PubMed Scopus (2009) Google Scholar). After incubation of HASMC with glucose, medium was aspirated, and lysis buffer (10 mm Tris-HCL, pH 7.5, 10 mm NaCl, 3 mm MgCl2, 0.5% Nonidet P-40, protease inhibitors) was added to the cells. All the buffers were prepared in diethyl pyrocarbonate-treated water. After shaking at 4 °C for 10 min, cells were scraped and spun for 5 min at 1000 × g at 4 °C, and the pellet was washed with lysis buffer. Nuclei were resuspended in storage buffer (100 μl/plate) (50 mm Tris pH 8.3, 40% glycerol, 5 mm MgCl2, 0.1 mm EDTA) and stored at -80 °C until used. For the in vitro transcription reactions, 20 million nuclei were resuspended in the reaction buffer (40 mm Tris, pH 8.5, 150 mm NH4Cl, 7.5 mm MgCl2, 0.62 mm ATP, 0.31 mm GTP, 0.31 mm CTP, 80 units of RNase inhibitor, 125 μCi of [32P]UTP) and incubated for 20 min at 27 °C. 3 μ l of 10 mm UTP was added, and the incubation was continued for 10 min. This step was followed by the addition of 2 μl of RNase-free DNase I (10 units/ml), and the incubation was further continued for 10 min at 27 °C. Then, 75 μl of 20% SDS, 15 μ l of 0.5 m EDTA, 5 μl of 1 m Tris-HCl, pH 7.4, and 55 μl of 10 mg/ml proteinase K were added, and the resulting mixture was incubated for 2 h at 42 °C. After phenol extraction and ethanol precipitation, RNA was dissolved in 50 μl of diethyl pyrocarbonate-treated water. 5 μg of TSP-1 or β-actin cDNA and 0.25 μg of 28 S cDNA were dissolved in a denaturing solution (25 μ l of 10 m NaOH, 4 m NaCl/ml), spotted on nylon membranes (PerkinElmer), UV-cross-linked, and dried in air. Membranes were prehybridized for 1 h at 42°C. The heat-denatured RNA probe and RNase inhibitor were added, and hybridization was performed at 42 °C overnight. After two 90-min washes in 30 mm sodium citrate, pH 7, 300 mm NaCl, 0.1% SDS at 42 °C, membranes were exposed to a Kodak BioMax MR film. Inhibition of Specific Metabolic Pathways—HASMC were incubated with 30 mm d-glucose for 24 h in the presence or absence of different glycosylation inhibitors (40 μm 6-diazo-5-oxonorleucine (DON), 40 μm azaserine, 1 mm benzyl 2-deoxy-α-d-galactopyranoside, BG). In the case of DON, cells were preincubated for 6-18 h prior to stimulation. To demonstrate that the effect of DON on TSP-1 expression was due to the inhibition of GFAT and the absence of the downstream metabolites of the hexosamine pathway, we used glucosamine to overcome DON inhibition. In these experiments cells were preincubated with 40 μm DON for 6 h followed by stimulation with 2 mm glucosamine for 24 h. Proliferation of Cultured Smooth Muscle Cells—HASMC were plated in 24-well clusters (Costar) (5000 cells/well) in 10% FBS, DMEM/F-12. 24 h later, the medium was changed to low glucose (5 mm) DMEM, and treatments were initiated as follows. After 4 h of pretreatment with inhibitors as indicated in the figure legends, 30 mm d-glucose, l-glucose, mannose, fructose, galactose, 2 mm glucosamine, 100 μm PUGNAc, and 0.8 μg/ml TSP-1 (Sigma) were added. TSP-1 was added both at the time of stimulation and again the next day to imitate continuous production of TSP-1 by sugar-stimulated cells. In some of these experiments, as shown in Fig. 8A, cells were treated with 2 μg/ml of anti-TSP-1 antibody (Clone 6.1, LabVision) or a control antibody against unrelated protein added at the time of stimulation and once again the next day. The control antibody did not affect HASMC proliferation (not shown). After 4 days of proliferation, plates were washed with phosphate-buffered saline, and the amount of cell DNA/well was measured using a CyQuant Cell proliferation assay kit (Molecular Probes). The time point was chosen because when the cells were plated at the density of 5000 cells/well (lower limit of the linear zone of detection in CyQuant assay), the difference in proliferation between glucose-stimulated and non-stimulated cells was greatest at 4 days. In another series of experiments, siRNA for TSP-1 and control siRNA (Ambion) were delivered to HASMC by nucleofection (Amaxa) according to the manufacturer’s protocol. Cell Transfection and Luciferase Assay—HASMC were plated in 24-well clusters (Costar) (0.3 × 106 cells/well) in 10% FBS, DMEM/F-12 media. The next day cells were transiently transfected with a -2033/+66 pTHBS-1 promoter-luciferase reporter construct, a control pGL3 vector, or with a -121/+66 pTHBS-1 promoter-luciferase construct, which was found to be unresponsive to glucose but still had high constitutive activity comparable with the full promoter reporter construct. The transfection procedure was carried out using Lipofectin reagent (Invitrogen) following the manufacturer’s protocol. 6 h post-transfection, the plasmid DNA-containing medium was changed to low glucose (5 mm) 10% FBS, DMEM, and the transfected cells were treated with 30 mm glucose, fructose, mannose, or galactose, as appropriate. For inhibitor studies, cells were incubated with 30 mm glucose in the presence or absence of different glycosylation inhibitors, as indicated in the figures. After 42 h of incubation, cell extracts were assayed for luciferase activity using a luciferase assay kit (Promega). Protein concentrations in cell lysates were analyzed using the BCA protein assay reagent (Pierce), and the activity of luciferase was normalized to total protein concentrations in lysates. Overexpression of Glutamine; Fructose 6-Phosphate Amidotransferase—HASMC were trypsinized, washed, and plated in 6-well clusters (Costar) (0.8 × 106 cells per well) in 10% FBS, DMEM/F-12 media. Confluent cells were transiently transfected with 2 μg GFAT-pcDNA3.1 or with pcDNA3.1 as a control in Opti-MEM-1 reduced serum medium using Lipofectin reagent (Invitrogen) according to the manufacturer’s protocol. 5 h post-transfection, the plasmid DNA-containing medium was replaced by 10% FBS, DMEM/F-12 media. Total RNA was extracted from the transfected cells 48 h post-transfection and subjected to Northern blotting, as previously described. To detect GFAT protein expression, whole cell lysates were prepared from the transfected cells using a hypotonic cell lysis buffer containing Nonidet P-40. Cell pellets were resuspended in protein sample buffer and boiled for 10 min, and samples were resolved in 8% SDS-PAGE gel. Proteins were visualized by staining with Coomassie Blue to detect overexpression of the 80-kDa protein. Western Blot—Lysates of cells transfected with GFAT or controls were analyzed by SDS gel electrophoresis, and Western blotting was performed using human GFAT antibody (36Weigert C. Friess U. Brodbeck K. Haring H.U. Schleicher E.D. Diabetologia. 2003; 46: 852-855Crossref PubMed Scopus (44) Google Scholar) kindly provided by Dr. E. D. Schleicher, University of Tubingen, Germany, anti-O-GlcNAc, an antibody recognizing cytoplasmic and intranuclear O-linked glycoproteins (Affinity Bioreagents, Inc.), and β-actin antibodies. Lysates of cells treated with different sugars, glucosamine, STZ, PUGNAc, and DON, as indicated in the figure legend, were resolved in 12% SDS-PAGE gel, and Western blotting was performed using anti-O-GlcNAc. The membrane was also stained with Ponceau S to determine equal protein loading. Detection of Intracellular Glycosylation by Immunofluorescence—Immunofluorescence was done using anti-O-GlcNAc antibody as described by Chen et al. (37Chen G. Liu P. Thurmond D.C. Elmendorf J.S. FEBS Lett. 2003; 534: 54-60Crossref PubMed Scopus (34) Google Scholar). HASMC were grown on coverslips in 6-well clusters (Costar; 0.75 × 106 cells per well) in 10% FBS, DMEM/F-12 medium. Confluent cells were placed in 0.2% FBS, low glucose (5 mm) DMEM for 24 h and then incubated with 30 mm d-glucose, mannose, and galactose for varying periods of time. At the appropriate end points, the media were aspirated, and the cells were fixed for 20 min at 25 °C in a solution containing 4% paraformaldehyde and 0.2% Triton-X. The cells were blocked in 5% donkey serum for 60 min at 25 °C. Subsequently, cells were incubated for 60 min at 25 °C with a 1:100 dilution of anti-O-GlcNAc (clone RL2) mouse monoclonal antibody against O-linked N-acetylglycosylated proteins in 5% donkey serum followed by incubation with a 1:50 dilution of rhodamine red-X-conjugated donkey anti-mouse immunoglobulin G (Jackson Immunoresearch Inc.) for 60 min at 25 °C. Quantitation of immunofluorescence was performed with Image-Pro Plus 4.5.1 (Media Cybernetics). Lactate Dehydrogenase Assay—The cytotoxicity of all sugars and inhibitors at the concentrations used was detected by the measurement of lactate dehydrogenase activity released from the cytosol of damaged cells into the supernatant, according to the manufacturer’s protocol (Roche Applied Science). Briefly, HASMC were grown in 6-well clusters in full growth medium. 24 h before an experiment, cells were placed in low glucose (5 mm) DMEM, 0.2% FBS and incubated with different glucose analogs in the presence or absence of glycosylation inhibitors, as previously described. At the appropriate end point, 100 μ l of supernatant was placed in a 96-well microtiter plate followed by the addition of 100 μl of the reaction mixture provided in the assay kit. The resulting mixture was incubated at room temperature for 30 min, and the absorbance of the samples was measured at 490 nm using a microplate reader. To determine the maximum lactate dehydrogenase release, the supernatant collected from Triton X-100-treated cells was included as a positive control. XTT (Sodium 3,3′-{1-[Phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro) Benzene Sulfonic Acid Hydrate) Cell Viability Assay—The toxicity of glycosylation inhibitors at concentrations used in the present study was also assessed by the XTT viability assay based on the measurement of the activity of mitochondrial enzymes, according to the manufacturer’s protocol (Biotium, Inc.). Briefly, HASMC were plated in 96-well tissue culture plates. Confluent cells were placed in 100 μl of low glucose (5 mm) DMEM, 0.2% FBS for 24 h before stimulation with sugars. Cells were then treated with different glucose analogs as well as with glycosylation and GFAT inhibitors, as previously described. At the appropriate end point, activated XTT solution was added to each well, incubation was continued for 2-5 h, and the absorbance of the samples was measured at 490 nm using a microplate reader. Statistical Analyses—All values are presented as the means ± S.E., and significant differences between means were evaluated by the paired Student’s t test. Probability values of ≤0.05 were considered to be statistically significant. Identification of the Hexosamine Pathway as a Candidate Mediator of TSP-1 Up-regulation by High Glucose in HASMC—Most of the effects of glucose depend on its intracellular breakdown (38Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7128) Google Scholar, 39Sheetz M.J. King G.L. J. Am. Med. Assoc. 2002; 288: 2579-2588Crossref PubMed Scopus (825) Google Scholar). However, some effects result from a change in osmolarity or cell membrane properties and do not require glucose uptake by cells (40Igarashi M. Wakasaki H. Takahara N. Ishii H. Jiang Z.Y. Yamauchi T. Kuboki K. Meier M. Rhodes C.J. King G.L. J. Clin. Investig. 1999; 103: 185-195Crossref PubMed Scopus (361) Google Scholar). To find out whether intracellular glucose metabolism is required for TSP-1 expression as well as to identify the specific steps in intracellular glucose breakdown controlling TSP-1 expression, we used different glucose analogs sharing distinct metabolic steps with glucose as follows; 1) glucose analogs that are transported into the cell and share limited metabolic steps with glucose (fructose, mannose, and galactose), 2) cell-permeable glucose analogs that cannot be metabolized (2-deoxyglucose, which cannot be metabolized beyond 2-deoxyglucose 6-phosphate), 3) a downstream metabolite of the glycolytic pathway (dihydroxyacetone), and 4) a cellimpermeable glucose analog to provide osmolarity control (biologically inactive l-glucose). 30 mm l-glucose did not affect TSP-1 mRNA levels, excluding osmolarity change as a cause of TSP-1 up-regulation (Fig. 1). 30 mm 2-deoxyglucose and dihydroxyacetone failed to induce TSP-1 expression, suggesting that a limited breakdown of glucose is required and sufficient for up-regulation of TSP-1 mRNA levels. Other sugars (30 mm fructose, mannose, and galactose) increased TSP-1 mRNA expression up to 4-fold as compared with control cells (Fig. 1). This effect is similar to that of 30 mm d-glucose, suggesting that these four sugars share the metabolic pathway controlling TSP-1 expression. Production of metabolites for protein glycosylation is the common pathway shared by all of these sugars (Fig. 2). In glucose metabolism, formation of metabolites of glycosylation is mediated by an activation of the hexosamine biosynthetic pathway. The hexosamine pathway starts with fructose 6-phosphate, and our results with 2-deoxyglucose and dihydroxyacetone also limited the relevant steps of glycolysis to this intermediate, suggesting the hexosamine pathway as a candidate for a metabolic pathway mediating TSP-1 up-regulation by high levels of glucose. Hexosamine Pathway and Protein Glycosylation Mediate the Increase in TSP-1 mRNA Levels—To confirm that the hexosamine pathway and protein glycosylation mediate the up-regulation of TSP-1, we used specific inhibitors of GFAT, the ratelimiting enzyme of the hexosamine pathway, and direct glycosylation inhibitors that interfered with the transfer of a sugar moiety to a protein. Preincubation of HASMC with GFAT inhibitors, DON (40 μm, 18 h), and azaserine (40 μm, added at the time of glucose stimulation) inhibited the increase in glucose-induced TSP-1 mRNA levels by up to 9-fold and up to 12.9-fold, respectively (Fig. 3, A and B), as quantified by densitometric scans of Northern blots. Similarly, when HASMC were preincubated with 30 mm glucose in the presence of the direct glycosylation inhibitor BG (1 mm, added at the time of stimulation with glucose), there was no increase in TSP-1 mRNA expression. As shown in Fig. 3C, TSP-1 mRNA expression in response to 30 mm glucose was inhibited by 3-fold. The inhibitors also down-regulated the basal level of TSP-1, suggesting that the physiological concentration of glucose maintains constitutive TSP-1 expression. However, this down-regulation was not statistically significant, as shown in Fig. 3 (lower panel, representative of 3-4 independent experiments). The toxicity of the inhibitors was assessed by the release of lactate dehydrogenase and by the activity of mitochondrial enzymes, as described under “Experimental Procedures.” Neither the viability nor the metabolic activity was affected by any of the inhibitors in the indicated concentrations for the times used in our experiments (data not shown). To confirm that the activation of the hexosamine pathway results in increased expression of TSP-1, we incubated HASMC for 24 h with 2 mm glucosamine, a downstream metabolite of the hexosamine pathway. In addition, cells were treated for 24 h with STZ (5 mm) and PUGNAc (100 μm). Both compounds are known to increase O-linked protein glycosylation by effectively inhibiting β-N-acetylglucosaminidase, an enzyme responsible for cleavage of O-GlcNAc residues from intracellular proteins. As shown in Fig. 3D, glucosamine, STZ, and PUGNAc significantly increased TSP-1 mRNA expression by 3.8-, 5.2-, and 5.2-fold, respectively, as compared with control cells. To show that the effect of GFAT inhibitors on TSP-1 expression is the result of specific inhibition of the hexosamine pathway and that the addition of downstream metabolites can overcome the effect of GFAT inhibitors, glucosamine was used to stimulate cells pretreated with DON. The inhibitory effect of DON (40 μm) on TSP-1 mRNA expression was completely reversed when cells were treated with glucosamine (Fig. 3E). Overexpression of GFAT Results in