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Glycated and carboxy-methylated proteins do not directly activate human vascular smooth muscle cells
Kidney International, Vol. 68 (2005), pp. 2756–2765
Glycated and carboxy-methylated proteins do not directly activate human vascular smooth muscle cells MANDY L. BALLINGER, MERLIN C. THOMAS, JULIE NIGRO, MELANIE E. IVEY, RODNEY J. DILLEY and PETER J. LITTLE Cell Biology of Diabetes Laboratory, Baker Heart Research Institute, Melbourne, The Alfred Hospital, Victoria, Australia; Department of Medicine, Monash University, Central and Eastern Clinical School, The Alfred Hospital, Melbourne, Victoria, Australia; Danielle Alberti Memorial Centre for Diabetes Complications, Baker Heart Research Institute, The Alfred Hospital, Melbourne, Victoria, Australia; and St. Vincent’s Hospital, Fitzroy, Melbourne, Australia
Glycated and carboxy-methylated proteins do not directly activate human vascular smooth muscle cells. Background. Advanced glycation end products (AGEs) accumulate in patients with diabetes, particularly at sites of vascular damage and within atherosclerotic lesions, but whether they have direct actions on vascular smooth muscle cells (VSMCs) is controversial. Methods. AGEs were constructed and characterized by protein content, level of modification, fluorescence, and molecular size. Human VSMCs were derived from different vascular beds. Glucose consumption, de novo protein synthesis, and proteoglycan biosynthesis were measured using a colorimetric assay and metabolic radiolabeling. Receptor for AGEs (RAGE) expression was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results. Treatment with AGEs under low or high glucose conditions showed no change in cellular glucose consumption or in cellular protein synthesis under low glucose conditions. Treatment of VSMCs with Ne-(carboxymethyl)lysine in the presence of low glucose increased [35 S]-sulfate incorporation into secreted proteoglycans by 72% (P < 0.001) and 67% (P < 0.001); however, the control proteins also increased [35 S]-sulfate incorporation into proteoglycans by 56% (P < 0.01), with similar effects observed under high glucose conditions. Human VSMCs showed no difference in response to glycated and nonglycated protein. Protein and gene expression of RAGE in VSMC was approximately 50-fold lower compared to HMEC-1 and U937 cells, consistent with the immunohistochemical staining of RAGE in vivo. Conclusion. VSMCs show very low levels of RAGE expression; thus, activation of VSMCs by AGEs does not occur. In diabetes, RAGE expression in VSM may increase to the extent that it becomes activated by AGEs in a manner that would contribute to the process of atherosclerosis.
Key words: advanced glycation end products, atherosclerosis, de e novo protein synthesis, fluorescence, glucose consumption, N (carboxymethyl)lysine, proteoglycans, RAGE, vascular smooth muscle. Received for publication July 15, 2004 and in revised form March 31, 2005, and June 22, 2005 Accepted for publication July 14, 2005 C
2005 by the International Society of Nephrology
Advanced glycation end products (AGEs) represent a heterogenous group of carbohydrate-protein adducts that have been recently implicated in the development and progression of atherosclerosis [1, 2]. Increasing age, dyslipidemia, oxidative stress, and hyperglycemia result in the enhanced accumulation of AGEs, particularly at sites of vascular injury [1, 2]. Inhibition of AGE accumulation is able to attenuate accelerated atherosclerosis associated with diabetes, without restoring glucose levels to the normal range [3, 4]. This suggests that AGEs are key mediators of vascular injury in diabetes. However, the mechanisms by which AGEs promote atherosclerosis have not been elucidated. Many of the deleterious actions of AGEs are thought to be mediated by activation of multispecific receptors and binding proteins [1, 5]. In particular, the receptor for AGEs (RAGE) is thought to play an important role in the induction of inflammation, proliferation, and fibrogenesis following exposure to AGEs. RAGEs have been identified in human atherosclerotic plaques [6–9] and are up-regulated in atherosclerotic lesions associated with experimental diabetes [5, 10]. Furthermore, the use of soluble RAGE to competitively block activation of tissue associated RAGE results in reduced atherosclerotic lesion formation [5]. The cellular target for action(s) of vascular AGEs also remains to be established. There is little or no information on the direct effects of AGEs on human VSMCs. One report has shown that RAGE ligands do not induce proliferation of human vascular smooth muscle cells (VSMCs) [11]. The lack of AGE response on vascular smooth muscle is surprising in view of the cental role of this tissue in atherogenesis [12]. This is a very important point in terms of identifying the role of AGEs in human atherosclerosis and, subsequently, the opportunities to intervene to attenuate the atherosclerotic process. We have addressed this question in our established model of nontransformed human vascular smooth muscle cells derived
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from human vessels [13]. We have evaluated the direct effects of various AGEs on three properties of VSMCs, namely, glucose utilization [14], de novo protein synthesis [15], and enhanced proteoglycan biosynthesis including glycosaminoglycan elongation [16], which represents indicators of cellular activation, a hypertrophic response, and an atherogenic response, respectively. We have characterized the AGEs and assessed the levels of RAGE expression in VSMCs. METHODS Reagents All chemicals were of the highest grade and obtained from Sigma Chemicals (St. Louis, MO, USA) unless otherwise stated. Goat anti-RAGE polyclonal immunoglobulin G (IgG) was from Chemicon (Temecula, CA, USA). Avidin-biotin-peroxidase complex was from Vector Laboratories (Burlingame, CA, USA). Anti-goat IgG labeled with horseradish peroxidase (HRP) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fetal bovine serum (FBS) was from CSL (Melbourne, Australia). AGE construction and characterization Bovine serum albumin (BSA)-AGE was prepared by long-term incubation (60 days) of BSA fraction V (1 mmol/L) with D-glucose (1 mol/L) in sodium phosphate buffer, under sterile aerobic conditions at 37◦ C [17]. Ne -(carboxymethyl)lysine (CML) with two degrees of glycation were constructed by sterile incubation of glyoxylic acid (1 mg, 10 mg) and sodium cyanoborohydride (2 mg, 19.8 mg) dissolved in 0.4 mol/L sodium phosphate buffer, with BSA (176 mg) for 24 hours, in a total reaction volume of 1 mL [18]. Control proteins were prepared as described for BSA-AGE with the omission of D-glucose, and CML with the omission of glyoxylic acid and sodium cyanoborohydride. Extensive dialysis of preparations was performed postincubation against Dulbecco’s phosphate-buffered saline (PBS) to remove non-protein bound entities. The BSA-AGE and CML preparations were assessed for protein content and free amino groups using the BCA assay and the TNBS assay [19], respectively. The fluorescent properties of BSA-AGE, CML, and protein controls (1 mg/mL) were measured with excitation at 350 nm, emission 360–500 nm on a scanning spectrofluorometer (Spex Industries, Edison, NJ, USA). The size and derivative formation of BSA-AGE, CML, and protein controls was assessed on a 4% to 13% linear gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Cell culture Discarded segments of the internal mammary artery (IMA), radial artery (RA), and saphenous vein (SV)
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were obtained from the Alfred Hospital Cardiac Surgery Unit (Alfred Hospital, Melbourne, Australia), with at least 2 different patients with each vessel type. The Alfred Hospital Human Ethics Committee approved the acquisition of the tissue. Human vascular smooth muscle cell cultures were established using the explant technique as previously described [13], stained positive for smooth muscle a-actin, and showed characteristic “hills and valleys” by phase contrast microscopy [20]. The media utilized for cell growth was low (5 mmol/L) glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, and for experiments both low and high (25 mmol/L) glucose DMEM with 10% FBS were used. All experiments were conducted on cells from passage 8 to 17 in 24 well plates (Becton Dickinson, Franklin Lakes, NJ, USA). Prior to all experiments cells were rendered quiescent by incubation in low glucose DMEM, 0.1% FBS for 48 hours unless otherwise stated. U937 cells were obtained from Dr V.T. Tkachuk, Cardiology Research Center, Moscow, Russia. U937 cells were maintained in RPMI, 10% heat-inactivated serum containing antibiotics at 37◦ C. U937 cells were rendered quiescent in RPMI zero serum for 24 hours prior to treatment. Human microvascular endothelial cells (HMEC1) were obtained from Dr. Karlheinz Peter, Baker Heart Research Institute, Melbourne, Australia. HMEC1 cells were maintained in MCDB131 media containing 10% FBS and supplemented with epidermal growth factor (1 lg/100mL), hydrocortisone (1 lg/500mL), Lglutamine, and antibiotics. Glucose utilization Aerobic glycolysis is the major pathway of glucose utilization in vascular smooth muscle [21]. Glucose consumption, which is coupled with lactate production, was measured as a general indicator of cellular activation [14]. Confluent and quiescent VSMCs were treated with BSAAGE (1-100 lg/mL), TGF-b1 (2 ng/mL), short-term and long-term BSA controls (30 lg/mL each), CML least and most glycated (30, 100 lg/mL), CML most glycated (100 lg/mL), with TGF-b1 (2 ng/mL) and incubated at 37◦ C for 24 hours. Treatments were performed in the presence of low (5 mmol/L) and high (25 mmol/L) glucose medium containing 0.1% FBS. To determine cellular glucose consumption, glucose concentrations in the medium were determined pre- and post-treatment using the photometric hexokinase/NADPH reaction, and lactate was analyzed using a colorimetric reaction with lactate oxidase and peroxidase. Reaction end points were detected using a COBAS FARA Analytical System (Roche, Basel, Switzerland). Cells from each well were collected after trypsin treatment and counted on a Z2 Particle & Size Analyzer (Coulter Corp., Miami, FL, USA). U937 cells were treated with BSA-AGE (100 lg/mL) or long-term BSA
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control (100 lg/mL) in RPMI zero serum for 24 hours at 37◦ C and glucose utilization assessed as described above.
De novo protein synthesis Activation of cellular protein synthesis may precede an increase in DNA synthesis in vascular cells treated with growth stimuli [15]. To investigate the synthesis of new proteins, confluent human VSMC on 24-well plates were rendered quiescent and treated with CML least and most glycated (100 lg/mL), BSA-AGE (100 lg/mL), shortterm and long-term BSA controls (100 lg/mL each), TGF-b1 (2 ng/mL), and BSA-AGE (100 lg/mL) with TGF-b1 (2 ng/mL) in low and high glucose DMEM, 0.1% FBS. Treated cells were incubated for 16 hours at 37◦ C. The treatment medium was aspirated and replaced with low glucose DMEM, 0.1% FBS (1 mL), followed by the addition of 10 lCi/mL [35 S]-methionine/cysteine (ICN Biomedicals, Irvine, CA, USA) for 4 hours at 37◦ C. The medium was aspirated and the cells were washed three times with PBS followed by incubation with 10% trichloroacetic acid (TCA) for 30 minutes on ice to precipitate the proteins. The TCA was aspirated dry followed by a second treatment with TCA. To solubilize the protein, 0.1% SDS in 0.1 mol/L NaOH was added to each well for 30 minutes at 37◦ C, with occasional agitation. Protein solutions were neutralized with 1 mol/L acetic acid (100 lL) and counted for [35 S] in a liquid scintillation counter (Packard, Meriden, CT, USA).
Proteoglycan biosynthesis Proteoglycan biosynthesis was evaluated as a measure of a specific atherogenic response [22, 23]. TGF-b1 stimulates proteoglycan synthesis in vascular SMCs [22, 24]. Human VSMCs were grown to confluence on 24well plates and serum deprived for 48 hours. Cells were treated with TGF-b1 (1 ng/mL), short-term BSA control (100 lg/mL), CML least and most glycated (100 lg/mL). Vascular SMCs were metabolically labeled with [35 S]sulfate (100 lCi/mL) for 24 hours at 37◦ C. Identically treated parallel plates were established without radiolabel to count cells. The medium was collected from each well and the cell layer harvested as previously described [25]. To quantitate total proteoglycan biosynthesis an aliquot (50 lL) of the medium and cell samples were applied to individual sections of chromatography paper (Whatman 3MM; Maidstone, UK). The paper was dried and extensively washed in a solution containing 1% cetylpyridinium chloride (CPC), and 50 mmol/L NaCl. After drying and addition of scintillation fluid (10 mL), individual papers were counted for [35 S] in a liquid scintillation counter.
Real-time quantitative RT-PCR for RAGE VSMCs were harvested, homogenized, and total RNA was isolated using Ultra-Turrax (Janke & Kunkel IKA, Labortechnik, Germany) in TRIZOL (Life Technologies, Inc., Gaithersburg, MD, USA). cDNA was synthesized with a reverse transcriptase reaction using a standard technique (SuperscriptTM First Strand Synthesis System for RT-PCR; Life Technologies, Inc.) as previously described [26]. To assess genomic DNA contamination, controls without reverse transcriptase were included. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for the quantitative determination of mRNA. Briefly, gene specific 5 -oligonucleotide corresponding to the human RAGE (5 -AGGAATGGA AAGGAGACCAAGTC), RAGE 3 -oligonucleotide primer (5 -CGTGAGTTCAGAGGCAGAATCTAC), and RAGE probe (FAM5 -TCTACCAGATTCCTGG GAAGCCAGAAATTG-TAMRA) were designed using the software program ‘Primer Express’ (PE Applied Biosystems, Foster City, CA, USA), and real-time PCR was carried out as previously described [26]. The level of expression of 18S was used as the endogenous control to ensure equal starting amounts of cDNA. Expression of RAGE gene by SMCs derived from the IMA was used as the calibrator with a given value of 1, with other groups compared with this calibrator.
Western blotting for RAGE To investigate the presence and size of immunoreactive RAGE proteins in whole cell lysates, a Western blot was performed. Human VSMCs were grown to confluence on 60 mm plates, serum deprived for 24 hours, rinsed with PBS, then harvested by gentle scraping after application of a buffer containing Triton X-100 (1%), NaCl (5 mol/L), TRIS pH 7.5 (1 mol/L), EDTA (500 mmol/L), EGTA pH 9.0 (250 lmol/L), NP-40 (0.5%), PMSF (40 mmol/L), leupeptin (1%), and pepstatin (1%). The lysate was sonicated, centrifuged (1000 rpm, 10 min, 4◦ C), the supernatant collected, and protein content determined using the BCA assay. Protein (50 lg) was separated on a 10% SDS-PAGE and molecular weight markers (Invitrogen Life Technologies, Carlsbad, CA, USA) were used to indicate size. Proteins were transferred electrophoretically onto a polyvinylidene fluoride (PVDF) membrane and nonspecific binding sites blocked by immersion of membrane into 5% skim milk (1 hour). The membrane was incubated with goat anti-RAGE polyclonal IgG (1:1000) at 4◦ C overnight. Following washing, the secondary antibody, anti-goat IgG labeled with HRP (Santa Cruz Biotechnology), was applied at 1:2000 for 1 hour. The Chemiluminescence Reagent Plus system (NEN Life Sciences, Boston, MA, USA) was used to detect sites of primary antibody binding.
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Fig. 1. BSA-AGE but not Ne -(carboxymethyl)lysine (CML) shows fluorescent properties. AGEs were prepared as described in Methods and assessed for fluorescence at excitation k350 nm and emission k390– 490 nm. Buffer (), short-term BSA control (), long-term BSA control (◦), BSA-AGE (•), and most glycated CML (). BSA-AGE exhibited fluorescence, while the most glycated CML and controls did not show this property. Each trace was determined three times or more.
In vivo localization of vascular RAGE by immunohistochemistry Representative frozen sections of human IMA were prepared on a cryostat, fixed in acetone, and stained to identify and localize RAGE by immunohistochemistry. Nonspecific binding was blocked with 10% normal horse serum. The primary antibody, goat anti-RAGE IgG (1:400), was applied followed by detection with an avidinbiotin-peroxidase complex (Vector Laboratories) in conjunction with 3,3 -diaminobenzidine (Dako, Glostrup, Denmark) for color development. Controls included a positive rat lung sample (which showed strong positive staining only in the presence of the primary antibody) and a negative control where the primary antibody was substituted with goat IgG. Counterstaining was performed with hematoxylin. Statistical analyses Data are presented as the mean and standard error of the mean. Data were analyzed using a one-way analysis of variance (ANOVA). Results were considered significant when the P value was less than 0.05, 0.01, and 0.001, as indicated in individual figures. RESULTS Characterization of AGEs The constructed AGEs, BSA-AGE, and CMLs were characterized for protein content and free amino groups. BSA-AGE had 86% less free amino groups than BSA control protein, indicating significant glycation had occurred. Least and most glycated CML preparations
Control (fresh protein) Fig. 2. Electrophoretic migration of AGEs by SDS-PAGE. AGEs were prepared as described in Methods and separated on a 4-13% SDSPAGE followed by staining with 0.025% Coomassie Blue. BSA-AGE and most glycated CML showed an increase in size relative to the BSA control proteins. Gel represents two separate experiments.
showed a 21% and 73% reduction, respectively, in the available amino groups. Spectrofluorimetric analyses of the AGEs revealed BSA-AGE to exhibit fluorescence, but the BSA control protein and the least and most glycated CML did not exhibit fluorescence (Fig. 1). The BSA-AGE showed a reduction in the electrophoretic migration, indicating an increase in size relative to the long-term BSA control (Fig. 2). The most glycated CML showed reduced electrophoretic migration, indicating an increase in size relative to the short-term BSA control (Fig. 2). Effect of AGEs on cellular glucose utilization Vascular SMCs utilize glucose in glycolysis to produce ATP when ATP is consumed during any cellular activation. To demonstrate that an alteration in glucose utilization is an appropriate indicator of cellular activation, VSMCs were treated with TGF-b1, a known activator and atherogenic stimulus for SMCs. As expected, human VSMCs treated with TGF-b1 (2 ng/mL) showed a 1.9-fold (P < 0.001) and 2.1-fold (P < 0.001) increase in cellular glucose utilization under low and high glucose conditions, respectively (Fig. 3A and B). Human VSMCs treated with BSA-AGE (1-100 lg/mL) showed no change in glucose utilization relative to the long-term BSA control regardless of whether the treatment was in low or high glucose medium (Fig. 3A and B). Treatment of VSMCs with least and most glycated CMLs (30-100 lg/mL) showed no change in glucose utilization relative to the short-term BSA control under both low and high glucose conditions (Fig. 4A and B). Cotreatment of VSMCs with TGF-b1 and CML (100 lg/mL) did not alter cellular glucose consumption compared to cells treated with TGF-b1 alone
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Fig. 3. BSA-AGE treatment of VSMCs does not alter cellular glucose utilization. Human VSMCs were treated with DMEM containing 0.1% serum (Con), TGF-b1 (2 ng/mL), long-term BSA control (BSA Con, 30 lg/mL), or BSA-AGE (1-100 lg/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Results are a mean ± SEM from three separate experiments. Significance indicated relative to basal control (∗∗∗ P < 0.001).
(Fig. 4A and B). Agonist effects on glucose consumption were mirrored as expected by simultaneous determination of lactate production as the product of glycolysis (data not shown). Effect of AGEs on de novo protein synthesis The de novo protein synthesis assay was another potential measure of general cellular responsiveness to AGE treatment. Under low glucose conditions, cellular response to CML least glycated and CML most glycated were increased relative to the control by 25% (P < 0.01) and 28% (P < 0.01), respectively, but was not changed relative to the short-term BSA control (Fig. 5A). The increase in de novo protein synthesis under low glu-
Con TGF BSA 30 100 30 100 100 Con + TGF CML least CML most glycated (µg/mL) glycated (µg/mL) Fig. 4. CML treatment of VSMCs does not alter cellular glucose utilization. Human VSMCs were treated with DMEM containing 0.1% FBS (Con), TGF-b1 (2 ng/mL), short-term BSA control (BSA Con, 30 lg/mL), CML least glycated (30, 100 lg/mL) or CML most glycated (30, 100 lg/mL), and CML (100 lg/mL) most glycated with TGF-b1 (2 ng/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Results are mean ± SEM from two separate experiments. Significance indicated relative to basal control (∗ P < 0.05, ∗∗∗ P < 0.001).
cose conditions was not due to glycation of proteins. Under high glucose conditions, VSMCs treated with AGEs showed no change in de novo protein synthesis compared to untreated cells (Fig. 5B). Effect of AGEs on proteoglycan biosynthesis Growth factor activation of VSMCs leads to stimulation of proteoglycan synthesis and, specifically, elongation of GAGs and increased binding to low-density lipoprotein (LDL) [27]. Treatment of VSMCs with TGFb1 (1 ng/mL) increased [35 S]-sulfate incorporation into proteoglycans by 80% (P < 0.001) and 164% (P < 0.001)
Ballinger et al: Protein glycation and smooth muscle activation
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B 500 400 300 200 100 0 Con TGF BSA BSA BSA CML CML Con -AGE Con Least Most LT ST Fig. 5. Treatment of VSMCs with CMLs increases de novo protein synthesis relative to basal but not relative to BSA control under low glucose conditions. Human VSMCs were treated with DMEM containing 0.1% FBS (Con), TGF-b1 (2 ng/mL), long-term BSA control (BSA Con LT, 30 lg/mL), BSA-AGE (100 lg/mL), short-term BSA control (BSA Con ST, 30 lg/mL), CML least glycated (100 lg/mL) or CML most glycated (100 lg/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Cells were metabolically labeled with [35 S]-met/cys (10 lCi/mL) for 4 hours prior to analyzing de novo protein. Results are a mean ± SEM from three separate experiments. Significance indicated relative to basal control (∗ P < 0.05, ∗∗ P < 0.01).
under low and high glucose conditions, respectively, compared to untreated cells (Fig. 6A and B) [22]. Human VSMCs treated with the short-term BSA control showed an increase in [35 S]-sulfate incorporation into proteoglycans by 56% (P < 0.01) and 69% (P < 0.001) under low and high glucose conditions, respectively, compared to untreated cells (Fig. 6A and B). Treatment of cells with least and most glycated CMLs showed that [35 S]-sulfate incorporation into proteoglycans was increased by 72% (P < 0.001) and 67% (P < 0.001), respectively, relative to the control under low glucose conditions. However, the extent of stimulation with the CMLs was not different
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from the short-term BSA control (Fig. 6A). Similar effects of CML treatment on SMC proteoglycan synthesis were observed under high glucose conditions (Fig. 6B). The cell-associated proteoglycans were assessed following treatment with CMLs and the results reflected and confirmed those observed in the media samples (data not shown). Vascular SMCs treated with CMLs showed no change in the electrophoretic migration of radiolabeled proteoglycans, biglycan and decorin, by SDS-PAGE whether the treatment was under low or high glucose conditions, indicating that the increase in [35 S]-sulfate incorporation into proteoglycans was not associated with a growth factor– like increase in glycosaminoglycan length (Fig. 6C and D). Gene and protein expression of RAGE in human VSMCs and IMA sections Gene and protein expression of the full-length tissue RAGE was assessed by RT-PCR and Western blotting, respectively (Fig. 7A and B). Due to the low level of expression of RAGE in cells derived from the IMA we also determined the expression of RAGE in cells derived from two distinct vessels, the saphenous vein and radial arteries [28]. There was little variability in RAGE expression between the VSMC types despite their different vascular sites of derivation. The constitutive level of expression in the three human VSMC types was markedly less than seen in either U937 monocytes or human endothelial cells. Overall expression of the RAGE gene was over 200-fold lower in VSMCs when compared to monocytes or HMEC-1 cells. Protein expression of RAGE was at least 50-fold lower in VSMCs compared to monocytes and human endothelial cells. To confirm that the difference in RAGE expression is manifest as a functional difference in signaling we measured the effect of BSA-AGE on glucose consumption by U937 cells by identical methods to those used earlier for the consumption of glucose in vascular smooth muscle cells. Treatment of U937 cells with BSA-AGE (100 lg/mL) increased glucose consumption by 53.1 ± 8.5% (P < 0.001) relative to control (Fig. 7C). In vascular sections obtained from human IMA, immunohistochemical staining for RAGE was strongest in the endothelium and focal sites within the intima. Only scanty and diffuse staining was observed in VSMCs in the media, with an overall level of staining barely above background (Fig. 8A). The IMA section that was not treated with the primary antibody showed negative staining for RAGE (Fig. 8B).
DISCUSSION AGEs are strongly implicated in the etiology of the accelerated atherosclerosis that accompanies diabetes
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[5]. Vascular SMCs are the central cellular participant in atherosclerosis but the demonstration of atherogenic responses by VSMC to AGEs is lacking [11]. We prepared and characterized several AGEs and examined their acute effects on the responses of human VSMC, which would indicate cellular activation and a role in atherogenesis; the presence of RAGE on the chosen cell type was also determined by both immunohistochemistry and Western blotting. However, the multiple glycated proteins used in this study either did not have any effect on VSMCs, nor did they stimulate any cellular response over and above the nonglycated protein control. We further showed that VSMCs both in vitro and in vivo show very low levels of RAGE expression and, thus, activation of VSM by AGEs may not occur. We also confirmed that AGE treatment of U937 cells, which express a high level of RAGE, resulted in activation of cellular metabolism assessed as increased glucose utilization. The clinical manifestations of atherosclerosis leading to cardiovascular disease and acute clinical syndromes actually have three major components. First, the lipid-dependent binding and retention of atherogenic lipoproteins by vascular extracellular matrix molecules, including proteoglycans [22, 29, 30]; second, the inflammatory process of monocyte adhesion to the vessel wall
Fig. 6. Treatment of VSMCs with CMLs increases [35 S]-sulfate incorporation into proteoglycans relative to basal but not relative to BSA control. Human VSMCs were treated with DMEM containing 0.1% FBS (Con), TGF-b1 (1 ng/mL), short-term BSA control (BSA Con, 100 lg/mL), CML least glycated (100 lg/mL) or CML most glycated (100 lg/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Cells were metabolically labeled with [35 S]-sulfate (100 lCi/mL) for 24 hours prior to analysis of secreted proteoglycans by the CPC precipitation assay. Results are a mean ± SEM from three separate experiments. Significance indicated relative to basal control (∗∗ P < 0.01, ∗∗∗ P < 0.001). Radiolabeled proteoglycans from SMCs treated with short-term BSA control (BSA-Con, 30 lg/mL), CML most glycated (100 lg/mL), CML least glycated (100 lg/mL), or DMEM containing 0.1% FBS (Con) under (C) low (5 mmol/L) or (D) high (25 mmol/L) glucose conditions, were separated by SDS-PAGE.
and subsequent macrophage proliferation [12, 31]. These major events come together to generate lipid laden foam cells that develop into the complex lesions of atherosclerosis. The third major process is the thrombotic occlusion accompanying plaque rupture, which leads to the acute clinical syndrome [32]. There is now clear evidence for the involvement of AGEs and their receptors at critical but undefined points in the process [6]. It has previously been suggested that chronic low level activation of vascular AGE receptors by circulating AGEs and locally derived AGE modifications leads to vascular dysfunction and atherogenesis [33]. We hypothesize that one factor determining the nonresponsiveness of normal human VSMCs to the effects of AGEs may be the low level of constitutive expression of AGE receptors and, in particular, RAGE. In our studies the level of expression of RAGE both in vitro and in vivo was considerably lower than observed in endothelial cells or monocytes. However, in response to metabolic states such as diabetes, dyslipidemia, uremia, and aging, the expression of RAGE may be enhanced, possibly due to a positive feedback stimulus from high levels of AGEs in these conditions [5]. Certainly, at sites of diabetes-associated vascular injury, there is a high level of RAGE expression that colocalizes with AGEs and other atherogenic
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Fig. 7. RAGE gene and protein expression correlates with AGE mediated cellular activation in vascular cells. Data show results from (A) real time quantitative RT-PCR and (B) Western blotting for RAGE mRNA and protein, respectively. (A) Three lines of SMCs (IMA, RA, and SV), U937 monocytes/macrophages (Mu), and HMEC-1 cells were analyzed for RAGE expression. (B) For Western blotting a positive control of lung tissue is shown. Immunoreactive RAGE proteins in the 40 to 60 kD size range were detected. IMA, internal mammary artery; SVC, saphenous vein cells; RA, radial artery; Mu, U937 monocytes/macrophages. (C) U937 cells were treated with RPMI zero serum and BSA-AGE (100 lg/mL) or long-term BSA control (BSA Con, 100 lg/mL) for 24 hours at 37◦ C. Results are mean ± SEM from three separate experiments conducted in triplicate. Significance indicated relative to basal control (∗∗ P < 0.01, ∗∗∗ P < 0.001).
mediators (e.g., CTGF [34] and MCP-1 [35]) that have been implicated in the development and progression of human and animal atherosclerotic plaques [10, 36]. Thus, factors may operate in vivo in the presence of diabetes to up-regulate RAGE, and this generates VSMCs that can show atherogenic responses to AGEs. It will be important to determine the factors that can up-regulate RAGE expression. It is possible that the short-term nature of our studies failed to detect a change that may have been observed at later time points. For example, Chang et al have shown that oxidized LDL stimulates proteoglycan biosynthesis in VSMCs only after 72 hours of exposure [37]. However, long duration experiments are not suited to cell culture models since factors such as alterations in cell phenotype [38] and nutrient depletion or secretory product accumu-
lation in the media may impact the results. Such experiments may also reveal secondary effects, such as those resulting from the release of growth factors like PDGF [39] or TGF-b [40], which are known to mediate SMC proliferation. VSMC proliferation is thought to be consequent upon activation of de novo protein synthesis and, thus, this latter assay was also used as an early marker of proliferation in our experiments. The absence of activation of protein synthesis strongly suggests that there would be no mitogenic response. In addition, the experiments conducted in the present report also had positive controls demonstrating that cellular activation is indeed possible. Exposure to weak or partially competent agonists or growth factors may produce short-term responses such as an increase in intracellular pH [41] that do not manifest in cellular
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Theaters at the Alfred Hospital for assistance with the acquisition of the vessels used to develop the cell cultures.
A
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Reprint requests to Prof. Peter J. Little, Head, Cell Biology of Diabetes Laboratory Baker Heart Research Institute, St. Kilda Rd Central, PO Box 6492, Melbourne, Victoria 8008, Australia. E-mail: [email protected]
REFERENCES B
Control IgG
Fig. 8. Immunohistochemical staining for RAGE in human vascular rings. (A) Frozen sections of human IMA were immunohistochemically stained with goat anti-RAGE IgG (1:400). (B) A negative control of the human IMA section where the primary antibody was substituted with goat IgG.
responses in the absence of further stimulation. We did not evaluate very short-term signaling responses such as an increase in NF-jB [5] because our aim was to determine if AGEs could act as bona fide agonists capable of generating full atherogenic cellular responses. CONCLUSION Our data do not impact on the role or importance of AGEs in atherogenesis based on the lack of AGE stimulation of VSMCs due to the very low expression of RAGE in these cells and tissues. Insofar as the role of VSMCs is concerned, factors which may activate RAGE expression on VSMCs may draw these cells into the atherosclerotic cascade. The high-level RAGE expression in endothelial cells and macrophages both in vitro and in vivo, together with their known responsiveness to physiologic AGEs, also implicates these cells as possible key intermediates between AGE accumulation and VSMC activation. Identifying and clarifying these processes as they occur in the different settings of atherosclerosis is essential for understanding and, in the long-term, ameliorating human atherosclerosis. Such findings will impact the likely pathway to therapeutic agents capable of interfering in this signaling process and preventing macrovascular disease in diabetes. ACKNOWLEDGMENTS Support was provided by the Alfred Hospital Foundation (P.J.L.) and Diabetes Australia Research Trust Grant (P.J.L.). We thank Prof. Franklin Rosenfeldt and the staff of the C.J. Officer Brown Cardiac
1. VLASSARA H, PALACE MR: Diabetes and advanced glycation endproducts. J Intern Med 251:87–101, 2002 2. COOPER ME, BONNET F, OLDfiELD M, JANDELEIT-DAHM K: Mechanisms of diabetic vasculopathy: An overview. Am J Hypertens 14:475–486, 2001 3. PANAGIOTOPOULOS S, O’BRIEN RC, BUCALA R, et al: Aminoguanidine has an anti-atherogenic effect in the cholesterol-fed rabbit. Atherosclerosis 136:125–131, 1998 4. WOLFFENBUTTEL BH, BOULANGER CM, CRIJNS FR, et al: Breakers of advanced glycation end products restore large artery properties in experimental diabetes. Proc Natl Acad Sci U S A 95:4630–4634, 1998 5. SCHMIDT AM, YAN SD, WAUTIER JL, STERN D: Activation of receptor for advanced glycation end products: A mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circ Res 84:489–497, 1999 6. BURKE AP, KOLODGIE FD, ZIESKE A, et al: Morphologic findings of coronary atherosclerotic plaques in diabetics. A postmortem study. Arterioscler Thromb Vasc Biol 24:1266–1271, 2004 7. STITT AW, HE C, FRIEDMAN S, et al: Elevated AGE-modified ApoB in sera of euglycemic, normolipidemic patients with atherosclerosis: relationship to tissue AGEs. Mol Med 3:617–627, 1997 8. PALINSKI W, KOSCHINSKY T, BUTLER SW, et al: Immunological evidence for the presence of advanced glycosylation end products in atherosclerotic lesions of euglycemic rabbits. Arterioscler Thromb Vasc Biol 15:571–582, 1995 9. KUME S, TAKEYA M, MORI T, et al: Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. Am J Pathol 147:654–667, 1995 10. SUN M, YOKOYAMA M, ISHIWATA T, ASANO G: Deposition of advanced glycation end products (AGE) and expression of the receptor for AGE in cardiovascular tissue of the diabetic rat. Int J Exp Pathol 79:207–222, 1998 11. RENARD CB, ASKARI B, SUZUKI LA, et al: Oleate, not ligands of the receptor for advanced glycation end-products, promotes proliferation of human arterial smooth muscle cells. Diabetologia 46:1676– 1687, 2003 12. ROSS R: Atherosclerosis—An inflammatory disease. N Engl J Med 340:115–126, 1999 13. NEYLON CB, LITTLE PJ, CRAGOE EJ, JR., BOBIK A: Intracellular pH in human arterial smooth muscle. Regulation by Na+/H+ exchange and a novel 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)- and HCO3(-)-dependent mechanism. Circ Res 67:814–825, 1990 14. POUYSSEGUR J, FRANCHI A, SILVESTRE P: Relationship between increased aerobic glycolysis and DNA synthesis initiation studied using glycolytic mutant fibroblasts. Nature 287:445–447, 1980 15. BOBIK A, GROOMS A, LITTLE PJ, et al: Ethyisopropylamiloridesensitive pH control mechanisms modulate vascular smooth muscle cell growth. Am J Physiol 260:C581–C588, 1991 16. WILLIAMS KJ, TABAS I: The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol 15:551–561, 1995 17. MAKITA Z, VLASSARA H, CERAMI A, BUCALA R: Immunochemical detection of advanced glycosylation end products in vivo. J Biol Chem 267:5133–5138, 1992 18. KISLINGER T, FU C, HUBER B, et al: N(epsilon)(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression. J Biol Chem 274:31740–31749, 1999 19. HABEEB AF: Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem 14:328–336, 1966
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20. SUDHIR K, HASHIMURA K, BOBIK A, et al: Mechanical strain stimulates a mitogenic response in coronary vascular smooth muscle cells via release of basic fibroblast growth factor. Am J Hypertens 14:1128–1134, 2001 21. SUZUKI LA, POOT M, GERRITY RG, BORNFELDT KE: Diabetes accelerates smooth muscle accumulation in lesions of atherosclerosis: Lack of direct growth-promoting effects of high glucose levels. Diabetes 50:851–860, 2001 22. LITTLE PJ, TANNOCK L, OLIN KL, et al: Proteoglycans synthesized by arterial smooth muscle cells in the presence of transforming growth factor-beta1 exhibit increased binding to LDLs. Arterioscler Thromb Vasc Biol 22:55–60, 2002 23. SCHONHERR E, JARVELAINEN HT, SANDELL LJ, WIGHT TN: Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem 266:17640– 17647, 1991 24. SCHONHERR E, JARVELAINEN HT, KINSELLA MG, et al: Plateletderived growth factor and transforming growth factor-beta 1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells. Arterioscler Thromb 13:1026–1036, 1993 25. NIGRO J, DILLEY RJ, LITTLE PJ: Differential effects of gemfibrozil on migration, proliferation and proteoglycan production in human vascular smooth muscle cells. Atherosclerosis 162:119–129, 2002 26. FORBES JM, YEE LT, THALLAS V, et al: Advanced glycation end product interventions reduce diabetes-accelerated atherosclerosis. Diabetes 53:1813–1823, 2004 27. BALLINGER M, NIGRO J, FRONTANILLA K, et al: Regulation of glycosaminoglycan structure and atherogenesis. Cell Mol Life Sci 61:1296–1306, 2004 28. DE DIOS ST, BRUEMMER D, DILLEY RJ, et al: Inhibitory activity of clinical thiazolidinedione peroxisome proliferator activating receptor-gamma ligands toward internal mammary artery, radial artery, and saphenous vein smooth muscle cell proliferation. Circulation 107:2548–2550, 2003 29. SKALEN K, GUSTAFSSON M, RYDBERG EK, et al: Subendothelial retention of atherogenic lipoproteins in early atherosclerosis. Nature 417:750–754, 2002
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30. CAMEJO G: The interaction of lipids and lipoproteins with the intercellular matrix of arterial tissue: Its possible role in atherogenesis. Adv Lipid Res 19:1–53, 1982 31. PLUTZKY J: Inflammatory pathways in atherosclerosis and acute coronary syndromes. Am J Cardiol 88:10K–15K, 2001 32. FALK E, FERNANDEZ-ORTIZ A: Role of thrombosis in atherosclerosis and its complications. Am J Cardiol 75:3B–11B, 1995 33. BASTA G, LAZZERINI G, MASSARO M, et al: Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: A mechanism for amplification of inflammatory responses. Circulation 105:816–822, 2002 34. CANDIDO R, JANDELEIT-DAHM KA, CAO Z, et al: Prevention of accelerated atherosclerosis by angiotensin-converting enzyme inhibition in diabetic apolipoprotein E-deficient mice. Circulation 106:246– 253, 2002 35. BIERHAUS A, SCHIEKOFER S, SCHWANINGER M, et al: Diabetesassociated sustained activation of the transcription factor nuclear factor-kappaB. Diabetes 50:2792–2808, 2001 36. SOULIS T, THALLAS V, YOUSSEF S, et al: Advanced glycation end products and their receptors co-localise in rat organs susceptible to diabetic microvascular injury. Diabetologia 40:619–628, 1997 37. CHANG MY, POTTER-PERIGO S, TSOI C, et al: Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties. J Biol Chem 275:4766–4773, 2000 38. CAMPBELL JH, CAMPBELL GR: Culture techniques and their applications to studies of vascular smooth muscle. Clin Sci 85:501–513, 1993 39. WILSON E, MAI Q, SUDHIR K, et al: Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J Cell Biol 123:741–747, 1993 40. SAKATA N, MENG J, TAKEBAYASHI S: Effects of advanced glycation end products on the proliferation and fibronectin production of smooth muscle cells. J Atheroscler Thromb 7:169–176, 2000 41. SARDET C, COUNILLON L, FRANCHI A, POUYSSEGUR J: Growth factors induce phosphorylation of the Na+/H+ antiporter, glycoprotein of 110 kD. Science 247:723–726, 1990
Glycated and carboxy-methylated proteins do not directly activate human vascular smooth muscle cells MANDY L. BALLINGER, MERLIN C. THOMAS, JULIE NIGRO, MELANIE E. IVEY, RODNEY J. DILLEY and PETER J. LITTLE Cell Biology of Diabetes Laboratory, Baker Heart Research Institute, Melbourne, The Alfred Hospital, Victoria, Australia; Department of Medicine, Monash University, Central and Eastern Clinical School, The Alfred Hospital, Melbourne, Victoria, Australia; Danielle Alberti Memorial Centre for Diabetes Complications, Baker Heart Research Institute, The Alfred Hospital, Melbourne, Victoria, Australia; and St. Vincent’s Hospital, Fitzroy, Melbourne, Australia
Glycated and carboxy-methylated proteins do not directly activate human vascular smooth muscle cells. Background. Advanced glycation end products (AGEs) accumulate in patients with diabetes, particularly at sites of vascular damage and within atherosclerotic lesions, but whether they have direct actions on vascular smooth muscle cells (VSMCs) is controversial. Methods. AGEs were constructed and characterized by protein content, level of modification, fluorescence, and molecular size. Human VSMCs were derived from different vascular beds. Glucose consumption, de novo protein synthesis, and proteoglycan biosynthesis were measured using a colorimetric assay and metabolic radiolabeling. Receptor for AGEs (RAGE) expression was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Results. Treatment with AGEs under low or high glucose conditions showed no change in cellular glucose consumption or in cellular protein synthesis under low glucose conditions. Treatment of VSMCs with Ne-(carboxymethyl)lysine in the presence of low glucose increased [35 S]-sulfate incorporation into secreted proteoglycans by 72% (P < 0.001) and 67% (P < 0.001); however, the control proteins also increased [35 S]-sulfate incorporation into proteoglycans by 56% (P < 0.01), with similar effects observed under high glucose conditions. Human VSMCs showed no difference in response to glycated and nonglycated protein. Protein and gene expression of RAGE in VSMC was approximately 50-fold lower compared to HMEC-1 and U937 cells, consistent with the immunohistochemical staining of RAGE in vivo. Conclusion. VSMCs show very low levels of RAGE expression; thus, activation of VSMCs by AGEs does not occur. In diabetes, RAGE expression in VSM may increase to the extent that it becomes activated by AGEs in a manner that would contribute to the process of atherosclerosis.
Key words: advanced glycation end products, atherosclerosis, de e novo protein synthesis, fluorescence, glucose consumption, N (carboxymethyl)lysine, proteoglycans, RAGE, vascular smooth muscle. Received for publication July 15, 2004 and in revised form March 31, 2005, and June 22, 2005 Accepted for publication July 14, 2005 C
2005 by the International Society of Nephrology
Advanced glycation end products (AGEs) represent a heterogenous group of carbohydrate-protein adducts that have been recently implicated in the development and progression of atherosclerosis [1, 2]. Increasing age, dyslipidemia, oxidative stress, and hyperglycemia result in the enhanced accumulation of AGEs, particularly at sites of vascular injury [1, 2]. Inhibition of AGE accumulation is able to attenuate accelerated atherosclerosis associated with diabetes, without restoring glucose levels to the normal range [3, 4]. This suggests that AGEs are key mediators of vascular injury in diabetes. However, the mechanisms by which AGEs promote atherosclerosis have not been elucidated. Many of the deleterious actions of AGEs are thought to be mediated by activation of multispecific receptors and binding proteins [1, 5]. In particular, the receptor for AGEs (RAGE) is thought to play an important role in the induction of inflammation, proliferation, and fibrogenesis following exposure to AGEs. RAGEs have been identified in human atherosclerotic plaques [6–9] and are up-regulated in atherosclerotic lesions associated with experimental diabetes [5, 10]. Furthermore, the use of soluble RAGE to competitively block activation of tissue associated RAGE results in reduced atherosclerotic lesion formation [5]. The cellular target for action(s) of vascular AGEs also remains to be established. There is little or no information on the direct effects of AGEs on human VSMCs. One report has shown that RAGE ligands do not induce proliferation of human vascular smooth muscle cells (VSMCs) [11]. The lack of AGE response on vascular smooth muscle is surprising in view of the cental role of this tissue in atherogenesis [12]. This is a very important point in terms of identifying the role of AGEs in human atherosclerosis and, subsequently, the opportunities to intervene to attenuate the atherosclerotic process. We have addressed this question in our established model of nontransformed human vascular smooth muscle cells derived
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from human vessels [13]. We have evaluated the direct effects of various AGEs on three properties of VSMCs, namely, glucose utilization [14], de novo protein synthesis [15], and enhanced proteoglycan biosynthesis including glycosaminoglycan elongation [16], which represents indicators of cellular activation, a hypertrophic response, and an atherogenic response, respectively. We have characterized the AGEs and assessed the levels of RAGE expression in VSMCs. METHODS Reagents All chemicals were of the highest grade and obtained from Sigma Chemicals (St. Louis, MO, USA) unless otherwise stated. Goat anti-RAGE polyclonal immunoglobulin G (IgG) was from Chemicon (Temecula, CA, USA). Avidin-biotin-peroxidase complex was from Vector Laboratories (Burlingame, CA, USA). Anti-goat IgG labeled with horseradish peroxidase (HRP) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fetal bovine serum (FBS) was from CSL (Melbourne, Australia). AGE construction and characterization Bovine serum albumin (BSA)-AGE was prepared by long-term incubation (60 days) of BSA fraction V (1 mmol/L) with D-glucose (1 mol/L) in sodium phosphate buffer, under sterile aerobic conditions at 37◦ C [17]. Ne -(carboxymethyl)lysine (CML) with two degrees of glycation were constructed by sterile incubation of glyoxylic acid (1 mg, 10 mg) and sodium cyanoborohydride (2 mg, 19.8 mg) dissolved in 0.4 mol/L sodium phosphate buffer, with BSA (176 mg) for 24 hours, in a total reaction volume of 1 mL [18]. Control proteins were prepared as described for BSA-AGE with the omission of D-glucose, and CML with the omission of glyoxylic acid and sodium cyanoborohydride. Extensive dialysis of preparations was performed postincubation against Dulbecco’s phosphate-buffered saline (PBS) to remove non-protein bound entities. The BSA-AGE and CML preparations were assessed for protein content and free amino groups using the BCA assay and the TNBS assay [19], respectively. The fluorescent properties of BSA-AGE, CML, and protein controls (1 mg/mL) were measured with excitation at 350 nm, emission 360–500 nm on a scanning spectrofluorometer (Spex Industries, Edison, NJ, USA). The size and derivative formation of BSA-AGE, CML, and protein controls was assessed on a 4% to 13% linear gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Cell culture Discarded segments of the internal mammary artery (IMA), radial artery (RA), and saphenous vein (SV)
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were obtained from the Alfred Hospital Cardiac Surgery Unit (Alfred Hospital, Melbourne, Australia), with at least 2 different patients with each vessel type. The Alfred Hospital Human Ethics Committee approved the acquisition of the tissue. Human vascular smooth muscle cell cultures were established using the explant technique as previously described [13], stained positive for smooth muscle a-actin, and showed characteristic “hills and valleys” by phase contrast microscopy [20]. The media utilized for cell growth was low (5 mmol/L) glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, and for experiments both low and high (25 mmol/L) glucose DMEM with 10% FBS were used. All experiments were conducted on cells from passage 8 to 17 in 24 well plates (Becton Dickinson, Franklin Lakes, NJ, USA). Prior to all experiments cells were rendered quiescent by incubation in low glucose DMEM, 0.1% FBS for 48 hours unless otherwise stated. U937 cells were obtained from Dr V.T. Tkachuk, Cardiology Research Center, Moscow, Russia. U937 cells were maintained in RPMI, 10% heat-inactivated serum containing antibiotics at 37◦ C. U937 cells were rendered quiescent in RPMI zero serum for 24 hours prior to treatment. Human microvascular endothelial cells (HMEC1) were obtained from Dr. Karlheinz Peter, Baker Heart Research Institute, Melbourne, Australia. HMEC1 cells were maintained in MCDB131 media containing 10% FBS and supplemented with epidermal growth factor (1 lg/100mL), hydrocortisone (1 lg/500mL), Lglutamine, and antibiotics. Glucose utilization Aerobic glycolysis is the major pathway of glucose utilization in vascular smooth muscle [21]. Glucose consumption, which is coupled with lactate production, was measured as a general indicator of cellular activation [14]. Confluent and quiescent VSMCs were treated with BSAAGE (1-100 lg/mL), TGF-b1 (2 ng/mL), short-term and long-term BSA controls (30 lg/mL each), CML least and most glycated (30, 100 lg/mL), CML most glycated (100 lg/mL), with TGF-b1 (2 ng/mL) and incubated at 37◦ C for 24 hours. Treatments were performed in the presence of low (5 mmol/L) and high (25 mmol/L) glucose medium containing 0.1% FBS. To determine cellular glucose consumption, glucose concentrations in the medium were determined pre- and post-treatment using the photometric hexokinase/NADPH reaction, and lactate was analyzed using a colorimetric reaction with lactate oxidase and peroxidase. Reaction end points were detected using a COBAS FARA Analytical System (Roche, Basel, Switzerland). Cells from each well were collected after trypsin treatment and counted on a Z2 Particle & Size Analyzer (Coulter Corp., Miami, FL, USA). U937 cells were treated with BSA-AGE (100 lg/mL) or long-term BSA
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control (100 lg/mL) in RPMI zero serum for 24 hours at 37◦ C and glucose utilization assessed as described above.
De novo protein synthesis Activation of cellular protein synthesis may precede an increase in DNA synthesis in vascular cells treated with growth stimuli [15]. To investigate the synthesis of new proteins, confluent human VSMC on 24-well plates were rendered quiescent and treated with CML least and most glycated (100 lg/mL), BSA-AGE (100 lg/mL), shortterm and long-term BSA controls (100 lg/mL each), TGF-b1 (2 ng/mL), and BSA-AGE (100 lg/mL) with TGF-b1 (2 ng/mL) in low and high glucose DMEM, 0.1% FBS. Treated cells were incubated for 16 hours at 37◦ C. The treatment medium was aspirated and replaced with low glucose DMEM, 0.1% FBS (1 mL), followed by the addition of 10 lCi/mL [35 S]-methionine/cysteine (ICN Biomedicals, Irvine, CA, USA) for 4 hours at 37◦ C. The medium was aspirated and the cells were washed three times with PBS followed by incubation with 10% trichloroacetic acid (TCA) for 30 minutes on ice to precipitate the proteins. The TCA was aspirated dry followed by a second treatment with TCA. To solubilize the protein, 0.1% SDS in 0.1 mol/L NaOH was added to each well for 30 minutes at 37◦ C, with occasional agitation. Protein solutions were neutralized with 1 mol/L acetic acid (100 lL) and counted for [35 S] in a liquid scintillation counter (Packard, Meriden, CT, USA).
Proteoglycan biosynthesis Proteoglycan biosynthesis was evaluated as a measure of a specific atherogenic response [22, 23]. TGF-b1 stimulates proteoglycan synthesis in vascular SMCs [22, 24]. Human VSMCs were grown to confluence on 24well plates and serum deprived for 48 hours. Cells were treated with TGF-b1 (1 ng/mL), short-term BSA control (100 lg/mL), CML least and most glycated (100 lg/mL). Vascular SMCs were metabolically labeled with [35 S]sulfate (100 lCi/mL) for 24 hours at 37◦ C. Identically treated parallel plates were established without radiolabel to count cells. The medium was collected from each well and the cell layer harvested as previously described [25]. To quantitate total proteoglycan biosynthesis an aliquot (50 lL) of the medium and cell samples were applied to individual sections of chromatography paper (Whatman 3MM; Maidstone, UK). The paper was dried and extensively washed in a solution containing 1% cetylpyridinium chloride (CPC), and 50 mmol/L NaCl. After drying and addition of scintillation fluid (10 mL), individual papers were counted for [35 S] in a liquid scintillation counter.
Real-time quantitative RT-PCR for RAGE VSMCs were harvested, homogenized, and total RNA was isolated using Ultra-Turrax (Janke & Kunkel IKA, Labortechnik, Germany) in TRIZOL (Life Technologies, Inc., Gaithersburg, MD, USA). cDNA was synthesized with a reverse transcriptase reaction using a standard technique (SuperscriptTM First Strand Synthesis System for RT-PCR; Life Technologies, Inc.) as previously described [26]. To assess genomic DNA contamination, controls without reverse transcriptase were included. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for the quantitative determination of mRNA. Briefly, gene specific 5 -oligonucleotide corresponding to the human RAGE (5 -AGGAATGGA AAGGAGACCAAGTC), RAGE 3 -oligonucleotide primer (5 -CGTGAGTTCAGAGGCAGAATCTAC), and RAGE probe (FAM5 -TCTACCAGATTCCTGG GAAGCCAGAAATTG-TAMRA) were designed using the software program ‘Primer Express’ (PE Applied Biosystems, Foster City, CA, USA), and real-time PCR was carried out as previously described [26]. The level of expression of 18S was used as the endogenous control to ensure equal starting amounts of cDNA. Expression of RAGE gene by SMCs derived from the IMA was used as the calibrator with a given value of 1, with other groups compared with this calibrator.
Western blotting for RAGE To investigate the presence and size of immunoreactive RAGE proteins in whole cell lysates, a Western blot was performed. Human VSMCs were grown to confluence on 60 mm plates, serum deprived for 24 hours, rinsed with PBS, then harvested by gentle scraping after application of a buffer containing Triton X-100 (1%), NaCl (5 mol/L), TRIS pH 7.5 (1 mol/L), EDTA (500 mmol/L), EGTA pH 9.0 (250 lmol/L), NP-40 (0.5%), PMSF (40 mmol/L), leupeptin (1%), and pepstatin (1%). The lysate was sonicated, centrifuged (1000 rpm, 10 min, 4◦ C), the supernatant collected, and protein content determined using the BCA assay. Protein (50 lg) was separated on a 10% SDS-PAGE and molecular weight markers (Invitrogen Life Technologies, Carlsbad, CA, USA) were used to indicate size. Proteins were transferred electrophoretically onto a polyvinylidene fluoride (PVDF) membrane and nonspecific binding sites blocked by immersion of membrane into 5% skim milk (1 hour). The membrane was incubated with goat anti-RAGE polyclonal IgG (1:1000) at 4◦ C overnight. Following washing, the secondary antibody, anti-goat IgG labeled with HRP (Santa Cruz Biotechnology), was applied at 1:2000 for 1 hour. The Chemiluminescence Reagent Plus system (NEN Life Sciences, Boston, MA, USA) was used to detect sites of primary antibody binding.
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Fig. 1. BSA-AGE but not Ne -(carboxymethyl)lysine (CML) shows fluorescent properties. AGEs were prepared as described in Methods and assessed for fluorescence at excitation k350 nm and emission k390– 490 nm. Buffer (), short-term BSA control (), long-term BSA control (◦), BSA-AGE (•), and most glycated CML (). BSA-AGE exhibited fluorescence, while the most glycated CML and controls did not show this property. Each trace was determined three times or more.
In vivo localization of vascular RAGE by immunohistochemistry Representative frozen sections of human IMA were prepared on a cryostat, fixed in acetone, and stained to identify and localize RAGE by immunohistochemistry. Nonspecific binding was blocked with 10% normal horse serum. The primary antibody, goat anti-RAGE IgG (1:400), was applied followed by detection with an avidinbiotin-peroxidase complex (Vector Laboratories) in conjunction with 3,3 -diaminobenzidine (Dako, Glostrup, Denmark) for color development. Controls included a positive rat lung sample (which showed strong positive staining only in the presence of the primary antibody) and a negative control where the primary antibody was substituted with goat IgG. Counterstaining was performed with hematoxylin. Statistical analyses Data are presented as the mean and standard error of the mean. Data were analyzed using a one-way analysis of variance (ANOVA). Results were considered significant when the P value was less than 0.05, 0.01, and 0.001, as indicated in individual figures. RESULTS Characterization of AGEs The constructed AGEs, BSA-AGE, and CMLs were characterized for protein content and free amino groups. BSA-AGE had 86% less free amino groups than BSA control protein, indicating significant glycation had occurred. Least and most glycated CML preparations
Control (fresh protein) Fig. 2. Electrophoretic migration of AGEs by SDS-PAGE. AGEs were prepared as described in Methods and separated on a 4-13% SDSPAGE followed by staining with 0.025% Coomassie Blue. BSA-AGE and most glycated CML showed an increase in size relative to the BSA control proteins. Gel represents two separate experiments.
showed a 21% and 73% reduction, respectively, in the available amino groups. Spectrofluorimetric analyses of the AGEs revealed BSA-AGE to exhibit fluorescence, but the BSA control protein and the least and most glycated CML did not exhibit fluorescence (Fig. 1). The BSA-AGE showed a reduction in the electrophoretic migration, indicating an increase in size relative to the long-term BSA control (Fig. 2). The most glycated CML showed reduced electrophoretic migration, indicating an increase in size relative to the short-term BSA control (Fig. 2). Effect of AGEs on cellular glucose utilization Vascular SMCs utilize glucose in glycolysis to produce ATP when ATP is consumed during any cellular activation. To demonstrate that an alteration in glucose utilization is an appropriate indicator of cellular activation, VSMCs were treated with TGF-b1, a known activator and atherogenic stimulus for SMCs. As expected, human VSMCs treated with TGF-b1 (2 ng/mL) showed a 1.9-fold (P < 0.001) and 2.1-fold (P < 0.001) increase in cellular glucose utilization under low and high glucose conditions, respectively (Fig. 3A and B). Human VSMCs treated with BSA-AGE (1-100 lg/mL) showed no change in glucose utilization relative to the long-term BSA control regardless of whether the treatment was in low or high glucose medium (Fig. 3A and B). Treatment of VSMCs with least and most glycated CMLs (30-100 lg/mL) showed no change in glucose utilization relative to the short-term BSA control under both low and high glucose conditions (Fig. 4A and B). Cotreatment of VSMCs with TGF-b1 and CML (100 lg/mL) did not alter cellular glucose consumption compared to cells treated with TGF-b1 alone
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Fig. 3. BSA-AGE treatment of VSMCs does not alter cellular glucose utilization. Human VSMCs were treated with DMEM containing 0.1% serum (Con), TGF-b1 (2 ng/mL), long-term BSA control (BSA Con, 30 lg/mL), or BSA-AGE (1-100 lg/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Results are a mean ± SEM from three separate experiments. Significance indicated relative to basal control (∗∗∗ P < 0.001).
(Fig. 4A and B). Agonist effects on glucose consumption were mirrored as expected by simultaneous determination of lactate production as the product of glycolysis (data not shown). Effect of AGEs on de novo protein synthesis The de novo protein synthesis assay was another potential measure of general cellular responsiveness to AGE treatment. Under low glucose conditions, cellular response to CML least glycated and CML most glycated were increased relative to the control by 25% (P < 0.01) and 28% (P < 0.01), respectively, but was not changed relative to the short-term BSA control (Fig. 5A). The increase in de novo protein synthesis under low glu-
Con TGF BSA 30 100 30 100 100 Con + TGF CML least CML most glycated (µg/mL) glycated (µg/mL) Fig. 4. CML treatment of VSMCs does not alter cellular glucose utilization. Human VSMCs were treated with DMEM containing 0.1% FBS (Con), TGF-b1 (2 ng/mL), short-term BSA control (BSA Con, 30 lg/mL), CML least glycated (30, 100 lg/mL) or CML most glycated (30, 100 lg/mL), and CML (100 lg/mL) most glycated with TGF-b1 (2 ng/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Results are mean ± SEM from two separate experiments. Significance indicated relative to basal control (∗ P < 0.05, ∗∗∗ P < 0.001).
cose conditions was not due to glycation of proteins. Under high glucose conditions, VSMCs treated with AGEs showed no change in de novo protein synthesis compared to untreated cells (Fig. 5B). Effect of AGEs on proteoglycan biosynthesis Growth factor activation of VSMCs leads to stimulation of proteoglycan synthesis and, specifically, elongation of GAGs and increased binding to low-density lipoprotein (LDL) [27]. Treatment of VSMCs with TGFb1 (1 ng/mL) increased [35 S]-sulfate incorporation into proteoglycans by 80% (P < 0.001) and 164% (P < 0.001)
Ballinger et al: Protein glycation and smooth muscle activation
[35S]-met/cys incorporation into de novo protein (cpm/well x 103)
A 500 *
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300 200 100 0 Con TGF BSA BSA BSA CML CML Con -AGE Con Least Most LT ST
[35S]-met/cys incorporation into de novo protein (cpm/well x 103)
B 500 400 300 200 100 0 Con TGF BSA BSA BSA CML CML Con -AGE Con Least Most LT ST Fig. 5. Treatment of VSMCs with CMLs increases de novo protein synthesis relative to basal but not relative to BSA control under low glucose conditions. Human VSMCs were treated with DMEM containing 0.1% FBS (Con), TGF-b1 (2 ng/mL), long-term BSA control (BSA Con LT, 30 lg/mL), BSA-AGE (100 lg/mL), short-term BSA control (BSA Con ST, 30 lg/mL), CML least glycated (100 lg/mL) or CML most glycated (100 lg/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Cells were metabolically labeled with [35 S]-met/cys (10 lCi/mL) for 4 hours prior to analyzing de novo protein. Results are a mean ± SEM from three separate experiments. Significance indicated relative to basal control (∗ P < 0.05, ∗∗ P < 0.01).
under low and high glucose conditions, respectively, compared to untreated cells (Fig. 6A and B) [22]. Human VSMCs treated with the short-term BSA control showed an increase in [35 S]-sulfate incorporation into proteoglycans by 56% (P < 0.01) and 69% (P < 0.001) under low and high glucose conditions, respectively, compared to untreated cells (Fig. 6A and B). Treatment of cells with least and most glycated CMLs showed that [35 S]-sulfate incorporation into proteoglycans was increased by 72% (P < 0.001) and 67% (P < 0.001), respectively, relative to the control under low glucose conditions. However, the extent of stimulation with the CMLs was not different
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from the short-term BSA control (Fig. 6A). Similar effects of CML treatment on SMC proteoglycan synthesis were observed under high glucose conditions (Fig. 6B). The cell-associated proteoglycans were assessed following treatment with CMLs and the results reflected and confirmed those observed in the media samples (data not shown). Vascular SMCs treated with CMLs showed no change in the electrophoretic migration of radiolabeled proteoglycans, biglycan and decorin, by SDS-PAGE whether the treatment was under low or high glucose conditions, indicating that the increase in [35 S]-sulfate incorporation into proteoglycans was not associated with a growth factor– like increase in glycosaminoglycan length (Fig. 6C and D). Gene and protein expression of RAGE in human VSMCs and IMA sections Gene and protein expression of the full-length tissue RAGE was assessed by RT-PCR and Western blotting, respectively (Fig. 7A and B). Due to the low level of expression of RAGE in cells derived from the IMA we also determined the expression of RAGE in cells derived from two distinct vessels, the saphenous vein and radial arteries [28]. There was little variability in RAGE expression between the VSMC types despite their different vascular sites of derivation. The constitutive level of expression in the three human VSMC types was markedly less than seen in either U937 monocytes or human endothelial cells. Overall expression of the RAGE gene was over 200-fold lower in VSMCs when compared to monocytes or HMEC-1 cells. Protein expression of RAGE was at least 50-fold lower in VSMCs compared to monocytes and human endothelial cells. To confirm that the difference in RAGE expression is manifest as a functional difference in signaling we measured the effect of BSA-AGE on glucose consumption by U937 cells by identical methods to those used earlier for the consumption of glucose in vascular smooth muscle cells. Treatment of U937 cells with BSA-AGE (100 lg/mL) increased glucose consumption by 53.1 ± 8.5% (P < 0.001) relative to control (Fig. 7C). In vascular sections obtained from human IMA, immunohistochemical staining for RAGE was strongest in the endothelium and focal sites within the intima. Only scanty and diffuse staining was observed in VSMCs in the media, with an overall level of staining barely above background (Fig. 8A). The IMA section that was not treated with the primary antibody showed negative staining for RAGE (Fig. 8B).
DISCUSSION AGEs are strongly implicated in the etiology of the accelerated atherosclerosis that accompanies diabetes
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C BSA-Con CML Most CML Least Con
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[5]. Vascular SMCs are the central cellular participant in atherosclerosis but the demonstration of atherogenic responses by VSMC to AGEs is lacking [11]. We prepared and characterized several AGEs and examined their acute effects on the responses of human VSMC, which would indicate cellular activation and a role in atherogenesis; the presence of RAGE on the chosen cell type was also determined by both immunohistochemistry and Western blotting. However, the multiple glycated proteins used in this study either did not have any effect on VSMCs, nor did they stimulate any cellular response over and above the nonglycated protein control. We further showed that VSMCs both in vitro and in vivo show very low levels of RAGE expression and, thus, activation of VSM by AGEs may not occur. We also confirmed that AGE treatment of U937 cells, which express a high level of RAGE, resulted in activation of cellular metabolism assessed as increased glucose utilization. The clinical manifestations of atherosclerosis leading to cardiovascular disease and acute clinical syndromes actually have three major components. First, the lipid-dependent binding and retention of atherogenic lipoproteins by vascular extracellular matrix molecules, including proteoglycans [22, 29, 30]; second, the inflammatory process of monocyte adhesion to the vessel wall
Fig. 6. Treatment of VSMCs with CMLs increases [35 S]-sulfate incorporation into proteoglycans relative to basal but not relative to BSA control. Human VSMCs were treated with DMEM containing 0.1% FBS (Con), TGF-b1 (1 ng/mL), short-term BSA control (BSA Con, 100 lg/mL), CML least glycated (100 lg/mL) or CML most glycated (100 lg/mL) under (A) low (5 mmol/L) or (B) high (25 mmol/L) glucose conditions. Cells were metabolically labeled with [35 S]-sulfate (100 lCi/mL) for 24 hours prior to analysis of secreted proteoglycans by the CPC precipitation assay. Results are a mean ± SEM from three separate experiments. Significance indicated relative to basal control (∗∗ P < 0.01, ∗∗∗ P < 0.001). Radiolabeled proteoglycans from SMCs treated with short-term BSA control (BSA-Con, 30 lg/mL), CML most glycated (100 lg/mL), CML least glycated (100 lg/mL), or DMEM containing 0.1% FBS (Con) under (C) low (5 mmol/L) or (D) high (25 mmol/L) glucose conditions, were separated by SDS-PAGE.
and subsequent macrophage proliferation [12, 31]. These major events come together to generate lipid laden foam cells that develop into the complex lesions of atherosclerosis. The third major process is the thrombotic occlusion accompanying plaque rupture, which leads to the acute clinical syndrome [32]. There is now clear evidence for the involvement of AGEs and their receptors at critical but undefined points in the process [6]. It has previously been suggested that chronic low level activation of vascular AGE receptors by circulating AGEs and locally derived AGE modifications leads to vascular dysfunction and atherogenesis [33]. We hypothesize that one factor determining the nonresponsiveness of normal human VSMCs to the effects of AGEs may be the low level of constitutive expression of AGE receptors and, in particular, RAGE. In our studies the level of expression of RAGE both in vitro and in vivo was considerably lower than observed in endothelial cells or monocytes. However, in response to metabolic states such as diabetes, dyslipidemia, uremia, and aging, the expression of RAGE may be enhanced, possibly due to a positive feedback stimulus from high levels of AGEs in these conditions [5]. Certainly, at sites of diabetes-associated vascular injury, there is a high level of RAGE expression that colocalizes with AGEs and other atherogenic
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Fig. 7. RAGE gene and protein expression correlates with AGE mediated cellular activation in vascular cells. Data show results from (A) real time quantitative RT-PCR and (B) Western blotting for RAGE mRNA and protein, respectively. (A) Three lines of SMCs (IMA, RA, and SV), U937 monocytes/macrophages (Mu), and HMEC-1 cells were analyzed for RAGE expression. (B) For Western blotting a positive control of lung tissue is shown. Immunoreactive RAGE proteins in the 40 to 60 kD size range were detected. IMA, internal mammary artery; SVC, saphenous vein cells; RA, radial artery; Mu, U937 monocytes/macrophages. (C) U937 cells were treated with RPMI zero serum and BSA-AGE (100 lg/mL) or long-term BSA control (BSA Con, 100 lg/mL) for 24 hours at 37◦ C. Results are mean ± SEM from three separate experiments conducted in triplicate. Significance indicated relative to basal control (∗∗ P < 0.01, ∗∗∗ P < 0.001).
mediators (e.g., CTGF [34] and MCP-1 [35]) that have been implicated in the development and progression of human and animal atherosclerotic plaques [10, 36]. Thus, factors may operate in vivo in the presence of diabetes to up-regulate RAGE, and this generates VSMCs that can show atherogenic responses to AGEs. It will be important to determine the factors that can up-regulate RAGE expression. It is possible that the short-term nature of our studies failed to detect a change that may have been observed at later time points. For example, Chang et al have shown that oxidized LDL stimulates proteoglycan biosynthesis in VSMCs only after 72 hours of exposure [37]. However, long duration experiments are not suited to cell culture models since factors such as alterations in cell phenotype [38] and nutrient depletion or secretory product accumu-
lation in the media may impact the results. Such experiments may also reveal secondary effects, such as those resulting from the release of growth factors like PDGF [39] or TGF-b [40], which are known to mediate SMC proliferation. VSMC proliferation is thought to be consequent upon activation of de novo protein synthesis and, thus, this latter assay was also used as an early marker of proliferation in our experiments. The absence of activation of protein synthesis strongly suggests that there would be no mitogenic response. In addition, the experiments conducted in the present report also had positive controls demonstrating that cellular activation is indeed possible. Exposure to weak or partially competent agonists or growth factors may produce short-term responses such as an increase in intracellular pH [41] that do not manifest in cellular
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Theaters at the Alfred Hospital for assistance with the acquisition of the vessels used to develop the cell cultures.
A
Anti-RAGE IgG
Reprint requests to Prof. Peter J. Little, Head, Cell Biology of Diabetes Laboratory Baker Heart Research Institute, St. Kilda Rd Central, PO Box 6492, Melbourne, Victoria 8008, Australia. E-mail: [email protected]
REFERENCES B
Control IgG
Fig. 8. Immunohistochemical staining for RAGE in human vascular rings. (A) Frozen sections of human IMA were immunohistochemically stained with goat anti-RAGE IgG (1:400). (B) A negative control of the human IMA section where the primary antibody was substituted with goat IgG.
responses in the absence of further stimulation. We did not evaluate very short-term signaling responses such as an increase in NF-jB [5] because our aim was to determine if AGEs could act as bona fide agonists capable of generating full atherogenic cellular responses. CONCLUSION Our data do not impact on the role or importance of AGEs in atherogenesis based on the lack of AGE stimulation of VSMCs due to the very low expression of RAGE in these cells and tissues. Insofar as the role of VSMCs is concerned, factors which may activate RAGE expression on VSMCs may draw these cells into the atherosclerotic cascade. The high-level RAGE expression in endothelial cells and macrophages both in vitro and in vivo, together with their known responsiveness to physiologic AGEs, also implicates these cells as possible key intermediates between AGE accumulation and VSMC activation. Identifying and clarifying these processes as they occur in the different settings of atherosclerosis is essential for understanding and, in the long-term, ameliorating human atherosclerosis. Such findings will impact the likely pathway to therapeutic agents capable of interfering in this signaling process and preventing macrovascular disease in diabetes. ACKNOWLEDGMENTS Support was provided by the Alfred Hospital Foundation (P.J.L.) and Diabetes Australia Research Trust Grant (P.J.L.). We thank Prof. Franklin Rosenfeldt and the staff of the C.J. Officer Brown Cardiac
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