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From the Laboratorio di Patologia Vascolare,*Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Roma; Dipartimento di Biologia,Università di Tor Vergata, Roma; Dipartimento di Scienza degli Alimenti,Università di Napoli "Federico II," Portici; and Consorzio Mario Negri Sud,S. Maria Imbaro, Chieti, Italy
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Materials and Methods |
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Primary bovine aortic endothelial cells (BAEC) were prepared and cultured as described.23 Endothelial cell culture purity was consistently >98% as determined by the cobblestone configuration of the endothelial cell monolayer and by the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) uptake (Biomedical Technologies Inc., Stoughton, MA, USA). All experiments were performed with cells at passages 2–6.
Cell Culture
Cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS) (Euroclone Inc., Milan, Italy), 2 mmol/L L-glutamine, and 100 IU/ml penicillin/streptomycin (Gibco BRL, Paisley, UK) in humidified 5% CO2 atmosphere at 37°C. The culture medium was changed every 3 or 4 days and cells were grown near confluence; cell monolayers were then harvested by exposure to 0.1% trypsin-EDTA (GIBCO BRL) as described.23
Migration Assay
Migration assays were carried out in modified Boyden chambers (Costar Scientific Corporation, Cambridge, MA, USA) as described.24 BAEC (2 x 105) were placed in the upper chamber of the Boyden apparatus mounted with 12-µm pores polycarbonate filters (Costar Scientific Corporation) coated with 5 mg/L gelatin solution (Sigma, St. Louis, MO, USA). Human recombinant FGF2 (Gibco BRL) was used as chemoattractant; it was dissolved at 10 ng/ml concentration in DMEM-0.1% BSA and placed in the lower chamber of the Boyden apparatus. For migration assays using unmodified or glycated FGF2 as chemoattractant, the growth factor at the end of glycation time was diluted with DMEM-0.1% BSA. Migration assays were carried out at 37°C in 5% CO2, for 5 hours, and then filters were removed, fixed with absolute ethanol, and stained with toluidine blue. Cells migrated were counted blindly at x400 magnification in 15 fields for each filter and the average number of cells/field was reported. All experiments were performed at least three times in triplicate.
Sugar-Dependent Modification of FGF2
Human recombinant FGF2 was dissolved in phosphate-buffered saline (PBS), pH 7.4, at 10 µg/ml final concentration and immediately frozen at –80°C. Then aliquots of FGF2 were incubated at 37°C for different times (up to 48 hours) with different concentrations (up to 250 mmol/L) of glucose, fructose, or mannitol, as described.21 These samples were tested in migration assays. FGF2 incubated with sugars was also subjected to Western blot and competitive ELISA analysis using a polyclonal antibody raised against glycated RNase or subjected to dot blot analysis with an anti-AGE monoclonal antibody (Dojindo). For radiolabeling studies, FGF2 (1 µg) was glycated in the presence of tracing amount of D-[1-14C]glucose or D-[U-14C]-fructose (2 µCi for each sugar) (Amersham Biosciences, Uppsala, Sweden; specific activity 57.0 mCi/mmol and 216 mCi/mmol for glucose and fructose, respectively), at 37°C, and electrophoresed by 8–15% gradient SDS-PAGE. Radiolabeled bands were detected after 5 days of exposure at -80°C on Kodak Optimax (Rochester, NY, USA) film.
Densitometry was carried out on a GS710 calibrated imaging densitometer (Bio-Rad, Hercules, CA, USA). To determine the number of AGE products/FGF2 molecules, the exact amount of FGF2 and labeled sugar within the bands were calculated by comparison with two calibration curves obtained by immunoblot and autoradiography of increasing amounts of FGF2 and labeled fructose, respectively.
Antibodies
Rabbit AGE-RNase polyclonal antibody was obtained against RNase (Sigma) incubated for 20 days with 25 mmol/L methyl glyoxal (MG) (Fluka, Buchs, Switzerland) according to published procedures.25 After incubation, the RNase-MG was dialyzed through a 12-KDa cut-off tube and used as antigen for rabbit immunization. Two 5-month-old New Zealand rabbits were immunized by injecting 0.5 mg of RNase-MG emulsified with complete Freund’s adjuvant (1:4 v:v). Rabbits received a booster injection 4 weeks after the first injection. Blood was collected 20 days after the last injection by ear puncture. The serum was separated and stored at -20°C until use.
Competitive ELISA
The antiserum was characterized by competitive ELISA as described.26 AGE-RNase antibody was assayed against different glycated proteins, ie, bovine serum albumin, ovalbumin, and casein, obtained by incubation with 250 mmol/L fructose for 7 days, or with ß-lactoglobulin incubated with 250 mmol/L methyl glyoxal for 7 days. For determination of FGF2 glycation, 96-well flexible ELISA plates were coated using a solution of 10 ng/ml glycated BSA (ie, preincubated with methyl glyoxal as described above) and incubated at 4°C overnight. After blocking with 10% horse serum in PBS, 0.1-ml aliquots of rabbit antiserum diluted 1:4000, in the presence of different amounts of glycated FGF2, were added at 37°C for 1 hour. Peroxidase-conjugated anti-rabbit IgG secondary antibodies and tetramethylbenzidine (Bio-Rad) were used for a colorimetric assay measuring OD at 450 nm.
Quantification of FGF2 Glycation
A competitive ELISA calibration curve was prepared using as reference a glycated ß-lactoglobulin, which bound an average of 4 mol of carbohydrates (AGE) per mole of protein. The relationship between percentage of inhibition and picomoles of AGE was given by the equation y = 17.4x + 7.06 (r2 = 0.9939).
The amount of AGE/µg of FGF2 under our conditions was then calculated by extrapolating the level of glycation from the calibration curve. The amount of AGE in ß-lactoglobulin was determined by electrospray mass spectrometry as described.11
Animals
Swiss (CD1) and non-obese diabetic NOD/LtJ mice were purchased from Charles River (Wilmington, MA). NOD mice have previously been shown to develop diabetes with clinical features similar to those of the human insulin-dependent diabetes mellitus, type I.27 Female mice, 18 to 19 weeks of age, were used for all experiments. Glucose blood levels were measured every 4 days after 6 hours of food starvation. Blood glucose levels were also measured immediately before starting angiogenesis assay and only NOD mice showing sugar levels higher than 200 mg/100 ml were considered hyperglycemic. Mice were housed under controlled temperature (23°C) and lighting with free access to water and standard mouse chow. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with international laws and policies (Guide for the Care and Use of Laboratory Animals. United States National Research Council, 1996).
Evaluation of in Vivo FGF2-Induced Angiogenesis
Angiogenesis in control and NOD mice was evaluated through the Matrigel assay.28 Briefly, a mixture of reconstituted basement membrane proteins (Matrigel, Beckton Dickinson, Franklin Lakes, NJ, 600 µl) was injected subcutaneously in NOD and CD1 mice (n = 6 for each group). Other groups were injected with the same mixture containing unmodified or glycated FGF2 (150 ng/ml). For these experiments, before mixing with Matrigel, FGF2 was incubated with 150 mmol/L mannitol, glucose, or fructose for 24 hours. To correlate glycemic levels with FGF2-induced angiogenesis, glucose blood level was measured in mice before angiogenesis assay was started. After 8 days from Matrigel injection, plugs were removed and processed for histology analysis. Histological sections (7 µm) were stained with Trichrome-Masson procedure (Bio-Optica, Milan, Italy). The vessels within the plugs were recognized by both morphology and presence of red blood cells. Angiogenesis was evaluated blindly by two operators, by considering at least four different sections per Matrigel plug; each section was 100 µm from the next. The total number of neovessels over the whole area of Matrigel was measured with a Quantimet 970 instrument (Cambridge Instruments, Cambridge, UK) and was expressed as number of vessels/mm2 as described.28 When two segments of vessels were cut longitudinally, as indicated by a long axis greater than threefold the short axis, and were close to each other, they were counted as a single vessel. In additional groups of mice injected with Matrigel containing unmodified or glycated FGF2, blood vessels in histological sections were identified by PECAM staining with an anti-PECAM-1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), according to manufacturer’s instructions.
FGF2 Glycation in Vivo
Recombinant FGF2 (2 µg per plug) was mixed with Matrigel (600 µl/mouse) and subcutaneously injected in control CD1 (n = 6) and NOD mice (n = 6). Control mice (n = 5) were injected with Matrigel alone. After 8 days, Matrigel plugs were removed, proteins within the plugs were extracted by homogenization with 10 strikes in a teflon/teflon potter system, in PBS containing the following inhibitors of proteases: 10 µmol/L aprotinin, 10 µmol/L leupeptin, 20 µmol/L pepstatin, 1 mmol/L benzamidine, 10 µmol/L PMSF (Sigma). After 30 minutes of centrifugation at 12,000 rpm at 4°C, supernatant was removed and immediately stored at –80°C. Proteins were analyzed by ELISA and Western blot. For ELISA analysis, 300 µg of protein extracted from Matrigel were loaded onto a plastic multiwell plate coated with a FGF2 monoclonal antibody; FGF2 glycation was detected with a monoclonal anti-AGE antibody (Dojindo Laboratories, Kumamoto, Japan) followed by a HRP-labeled secondary antibody and colorimetric assay. For Western blot analysis, total proteins extracted from Matrigel were subjected to immunoprecipitation with Sepharose-protein A beads (Pharmacia) activated with a polyclonal serum anti-FGF2 (Amersham) according to standard protocols and the manufacturer’s instructions. Equal amounts of FGF2 (85 ng as assessed by using a commercial ELISA assay for FGF2, R&D Systems, Minneapolis, MN, USA) were electrophoresed by SDS-PAGE and then electroblotted onto nitrocellulose membrane. Glycated proteins were detected by means of a polyclonal antibody raised against glycated RNase followed by ECL detection (Amersham).
Molecular Size Fractionation of FGF2 Gycation in Matrigel Plugs
To elucidate whether in vivo glycation involves the extracellular matrix-bound pool of FGF2 or the unbound pool, a fractionation study was carried out. Two groups of 5 hyperglycemic NOD mice each with blood glucose levels higher than 400 mg/100 ml were injected subcutaneously with Matrigel (600 µl) containing recombinant FGF2 (15 µg/plug). After 48 hours, plugs were removed and homogenized either in the presence of PBS (group A) or in the presence of 2 mol/L NaCl (group B). High ionic strength was used to detach FGF2 from ECM components. The homogenates were then subjected to 5 minutes of 1500-rpm microfuge centrifugation to remove large insoluble particles and the cleared supernatant was considered total homogenate. Both total homogenates from groups A and B were then subjected to 30 minutes at 14,000 rpm centrifugation. The supernatants were removed and subsequently ultrafiltrated through a 30 kd cut-off Millipore filter and proteins with molecular size higher or lower than 30 KDa were collected. The pellets from the centrifugation steps were not assayed due to the insolubility of samples. Each sample at different steps of fractionation was then immunoprecipitated by Sepharose-protein A-conjugated beads preactivated with an anti-FGF2 antibody (Santa Cruz) then assayed as reported above to quantify both FGF2 and glycation levels. The ultrafiltration allowed to identify two pools of FGF2: the first one with molecular size higher than 30 kd, which represents the high molecular weight (HMW) complexes, likely due to the binding of FGF2 to heparan sulfates and other ECM components. The second one, with molecular size lower than 30 kd, represents the low molecular weight (LMW) unbound pool of FGF2. The recovery of the injected FGF2 was not complete, probably due to diffusion of FGF2 out of the plugs, formation of stable insoluble complexes resistant to the high ionic strength treatment, stickiness of FGF2, and its attachment to the Millipore filters.
Statistical Analysis
Data were expressed as means ± SD. Student’s two-tailed t-test was performed and a P 
0.05 was considered statistically significant. Linear regression fitting of data were carried out with statistical software (SigmaStat for Windows, Jandel Scientific, Chicago, IL, USA).
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Results |
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To evaluate the effect of hyperglycemia on FGF2-induced angiogenesis, FGF2 was assayed in normoglycemic and hyperglycemic mice. CD1 control mice (n = 10) with blood sugar concentrations <100 mg/100 ml and NOD mice (n = 6) with blood sugar concentrations between 200 and 550 mg/100 ml were injected subcutaneously with 600 µl of Matrigel mixed with FGF2 (150 ng/ml). After 8 days, the angiogenic response within Matrigel plugs was determined in histological sections either by Trichrome-Masson staining (Figure 1A)
or by PECAM-1 immunostaining (Figure 1B)
. Results were similar regardless of which of these techniques was used to identify neoformed blood vessels. The angiogenic response to FGF2 was markedly reduced in diabetic mice as compared to controls (Figure 1C
, P < 0.001). Under similar experimental conditions, it was determined whether the impaired angiogenesis was directly related to the blood sugar level; the angiogenic response was assayed in NOD mice (n = 16) with blood glucose levels ranging between 50 and 550 mg/100 ml. After 8 days, mice were sacrificed and FGF2-induced angiogenesis was quantified and reported as a function of blood glucose level (Figure 1D)
. These experiments indicate that angiogenesis impairment in NOD mice was directly related to the blood sugar level (r = 0.81, P < 0.005). Since glucose is known to induce non-enzymatic glycation of proteins, we hypothesized that FGF2 glycation may occur in diabetic mice. To test this hypothesis, a polyclonal antibody able to recognize glycated proteins was developed and an ELISA assay to measure glycated FGF2 was set up.
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A competitive ELISA was set up with a polyclonal anti-AGE antibody developed against methyl glioxal-modified RNase. In this assay, the amount of glycated products is proportional to the competition measured. The antibody recognizes different glycated proteins and the competitive ELISA was 50% inhibited by 10 ng of AGE-BSA, 100 ng of AGE-caseine, 50 ng of AGE-ovalbumin, or 7 ng of ß-lactoglobulin (Figure 2)
. Further, anti-AGE antibody did not recognize an early glycation product such as fructosyl lysine and poor competition was observed with the dimer of methyl glioxal lysine (MOLD). Similar results were also obtained using this anti-AGE antibody in Western blotting assay (data not shown). Glycated ß-lactoglobulin, whose exact AGE number per molecule has been previously determined by electrospray mass spectrometry, was used as reference to draw a calibration curve. The linear fitting of such curve (r2 = 0.9939) (Figure 2
, inset) was used to calculate the picomoles of AGE/pmol of FGF2 by competitive ELISA (Table 1)
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In the following experiments, the ability of sugars to glycate FGF2 was investigated in vitro. To evaluate the extent of FGF2 glycation, a competitive ELISA using the anti-AGE polyclonal antibody was performed. In this assay, the level of protein glycation is directly related the inhibition measured. As shown in Table 1
, the amount of recombinant FGF2 glycation increased with time and with sugar concentration and was detectable after 48 hours exposure to 50 mmol/L fructose. In other experiments, recombinant FGF2 was incubated in the absence or in the presence of mannitol, glucose, or fructose (250 mmol/L for 24 hours). Samples were then subjected to immunoblot, followed by densitometric analysis (Figure 3A)
or SDS-PAGE under denaturing conditions, followed by Western blot analysis with anti-AGE polyclonal antibody (Figure 3B)
. Both analyses confirmed the formation of glycated-FGF2 after incubation with glucose or fructose. The glycation occurring in our experimental conditions did not modify significantly the antigenic properties of FGF2, as assessed by ELISA for FGF2 and in immunoprecipitation assays (not shown).
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. It was found that FGF2 was covalently modified by glucose and, more markedly, by fructose. Radiolabeled bands of FGF2 treated with labeled fructose (Figure 3C)
were then evaluated by densitometric quantification, which showed that after 48 hours incubation with fructose, about 1.1–1.8 glycation product(s) per FGF2 molecule had been formed. These data indicate that, under our experimental conditions, FGF2 can be glycated in vitro. Effect of Glycation on FGF2 Chemoattractant Properties
To evaluate the effect of glycation on FGF2 activity, BAEC migration assays were performed using either modified or unmodified FGF2 (10 ng/ml) as chemoattractant (Figure 4)
. These experiments showed that incubating FGF2 with glucose or fructose (50 or 150 mmol/L) for 24 hours led to a significant reduction of its chemoattractant properties, as compared to mannitol-treated FGF2 (P < 0.05 for glucose- and <0.01 for fructose-modified FGF2 versus control FGF2). These results further support the hypothesis that glycation may modify FGF2 angiogenic effects in vivo.
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The in vivo occurrence of FGF2 glycation was evaluated in hyperglycemic NOD mice using both monoclonal (Dojindo) and polyclonal anti-AGE antibodies. FGF2 (2 µg per plug) was mixed with Matrigel and injected subcutaneously in normoglycemic CD1 (n = 6) and hyperglycemic NOD (n = 6) mice. The blood sugar levels of CD1 and NOD mice were lower than 81 mg/100 ml (mean 64 ± 11 mg/100 ml) and higher than 305 mg/100 ml (mean 510 ± 92 mg/100 ml), respectively. After 8 days, Matrigel plugs were removed and the presence of glycated-FGF2 in both groups was evaluated by direct ELISA using a monoclonal anti-AGE antibody. To calculate the amount of AGE, both glycated ß-lactoglobulin and glycated BSA were used as standard, with similar results (not shown). FGF2 glycation was enhanced fourfold in hyperglycemic versus normoglycemic mice (P < 0.001) (Figure 5A)
. Similar results were obtained in this ELISA assay using the polyclonal anti-AGE-RNase serum (not shown). FGF2 glycation was confirmed by Western blot analysis using the polyclonal anti-AGE-RNase antibody (Figure 5B
, upper panel); in fact, a faint immunolabeling with the anti-AGE serum was detected with FGF2 immunoprecipitated from the plugs kept in normoglycemic mice, while a strong immunolabeling was detected with FGF2 immunoprecipitated from NOD mice. Densitometric analysis of the bands indicated a 9.4 fold increase of FGF2 glycation in NOD versus control CD1 mice (P < 0.001). Similar amounts of FGF2 were loaded in each lane (Figure 5B
, lower panel). Endogenous murine FGF2 was not detected in control Matrigel, ie, not containing recombinant FGF2, either by ELISA (not shown) or by Western blot (Figure 5B
, lower panel). This was likely due to the low amount of endogenous FGF2 in Matrigel plugs and to the inability of antibodies used in ELISA and in Western blot assays to recognize murine FGF2. These experiments demonstrate that enhanced FGF2 glycation occurred in hyperglycemic mice. Since FGF2, similarly to other heparin-binding growth factors, can bind heparan sulfates proteoglycans in the extracellular matrix (ECM), in additional experiments it was determined whether FGF2 glycation in vivo involved either the pool of FGF bound to the ECM components, or the pool of unbound FGF2, or both. To address this issue, hyperglycemic NOD mice were injected with Matrigel containing recombinant FGF2. After 48 hours, the Matrigel plugs were removed and a size fractionation study was carried out to evaluate the amount of glycated FGF2 present in the high molecular weight (HMW), ie, likely ECM-bound, and in the low molecular weight (LMW) complexes, ie, the unbound pool of FGF2. The results of the above experiments are summarized in Table 2
. These experiments indicate that in vivo glycation of FGF2 involves both pools of FGF2 and that the LMW unbound form shows a three- to fourfold higher number of AGE/FGF2 molecule than the HMW form (P < 0.05). To test whether FGF2 glycation may be related to the observed impairment of angiogenesis in diabetes, we evaluated the in vivo angiogenic properties of glycated FGF2.
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The angiogenic effect of glycated FGF2 versus unglycated FGF2 was tested in CD1 mice. The animals were injected with Matrigel mixed with FGF2 previously incubated for 24 hours at 37°C with 150 mmol/L glucose, fructose, or mannitol. Control mice were injected with Matrigel alone or with Matrigel containing FGF2 preincubated with 150 mmol/L mannitol or FGF2 supplemented with 150 mmol/L glucose or fructose immediately before the injection. After 8 days, Matrigel plugs were removed and the formation of new vessels was measured (Figure 6A)
. Under these conditions, FGF2 glycation in vitro inhibited the angiogenic effect of this growth factor in vivo. Microphotographs of histological sections from mice treated with unmodified and glycated FGF2 are shown in Figure 6B
(Trichrome Masson staining) and 6C (PECAM-1 immunostaining). It is noteworthy that angiogenesis induced by unmodified FGF2 was more marked in the experiments shown in Figure 1B
versus Figure 6A
. This discrepancy is likely related to temperature- and time-dependent loss of angiogenic activity due to the 24 hours incubation at 37°C of FGF2 with sugars. This result strongly support the hypothesis that the impairment of angiogenesis observed in NOD mice may be due, at least in part, to FGF2 glycation.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Glycation is a non-enzymatic reaction involving mainly the arginine and lysine residues in proteins.7
Protein glycation has been extensively studied in diabetes mellitus, and it leads to the formation of early, intermediate and advanced glycation products (AGEs).6,7
The role of AGEs in diabetic vasculopathy has been studied, and these products have been shown to promote a number of biochemical, cellular and pathophysiologic abnormalities, characteristic of diabetic vasculopathy.29
Presently it is still unclear whether AGEs are the cause of the vascular complications observed in patients suffering from diabetes mellitus.30
The effects of most AGEs are mediated through a receptor-dependent pathway requiring the interaction with a specific receptor for AGE (RAGE) leading to a cascade of intracellular effects.31
On the other hand, due to the high chemical reactivity of lysine and arginine residues, these amino acids play a key role in several posttranslational modifications, like methylation, transglutamination, myristoylation, proteolytic cleavage and protein-protein and protein-DNA interactions.32
Therefore, the chemical modification of such residue(s) due to glycation may lead to serious functional defects. Depending on the proteic target of glycation, hyperglycemia may induce at least two different effects: 1) chemical modifications of structural proteins like albumin, collagen, crystallins, and others, leading to a change of chemical-physical properties and therefore a change in solubility and aggregate formation (this process is related to the development of microangiopathy12
and to cataract formation8
in diabetes mellitus) and 2) chemical modifications of regulatory proteins possibly leading to functional defects (for instance, it has been shown an increase of AGE-modified low density lipoprotein (LDL) in plasma of diabetic patients, leading to a significant impairment of LDL-receptor-mediated clearance).33
FGF2 also may undergo such posttranslational modification: in fact, in vitro glycation of FGF2 has been shown to reduce its in vitro mitogenic activity.21
That study was mainly focused on intracellular FGF2 glycation, while a more recent report was focused on the glycation of extracellular FGF2.22
These in vitro studies suggested that FGF2 glycation may interfere with FGF2 binding to its receptor. On the other hand, inhibiting the methylation-dependent modification of FGF2 results in a significant decrease in FGF2 nuclear accumulation, suggesting that methylation as well as a correct structural conformation are relevant to the intracellular distribution of this growth factor.34
Although non-enzymatic glycation is generally considered a rather aspecific reaction, potentially involving any protein, it is noteworthy that only a few lysine or arginine residues in proteins are actually modified. In fact, the number of residues modified by fructose per FGF2 molecule was shown to range between 1.6 and 2.4 in a previous report21
and 1.1–1.8 in the present study, according to densitometric quantification of labeled FGF2 incubated with 14C-labeled sugars or between 0.8 and 3.9 pmol of AGE/pmol of FGF2 according to the results of the in vivo glycation assay reported in Table 2
. Human FGF2 contains 14 lysine and 11 arginine residues per molecule, therefore, according to these findings, only a few residues per FGF2 molecule are glycated, while the remaining lysine and arginine residues are unaffected. This suggests that some residues within FGF2 are more susceptible to glycation than others. Studies currently ongoing in our laboratory are aimed at the biochemical/functional characterization of this specificity.
In the present study endogenous FGF2 glycation could not be measured for several reasons: 1) very low serum levels of FGF2; 2) lack of good antibodies or ELISA kits specific for murine FGF2; and 3) high background from other glycated proteins. Therefore, in the present study, human recombinant FGF2 mixed with Matrigel was used to evaluate the amount of glycated FGF2. Similarly, since FGF2 serum levels in humans are in the picogram/ml range and the analytical methods developed in the present study are effective in the range of 10 to 100 ng of purified proteins/ml, it will be necessary to develop more sensitive assays to determine whether glycated FGF2 is present in human diabetic patients. It is also noteworthy that the competitive ELISA carried out in the present study and the antibodies used in Western blot experiments recognizes only advanced glycation products and may not reveal earlier glycation of FGF2 which is also likely to occur. In conclusion, our results suggest that in vivo FGF2 glycation may underlie the reduced angiogenic response to ischemia, observed in diabetic patients3,35 and in animal models1 of diabetes mellitus.
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Acknowledgements |
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Footnotes |
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Supported by Telethon Italia grants A61 and 1167.
Accepted for publication May 2, 2002.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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)-(carboxymethyl)lysine in human tissues in diabetes and aging. J Clin Invest 1997, 99:457-468[Medline]
-(carboxymethyl)lysine is increased in serum from children and adolescents with type 1 diabetes. Diabetes Care 1998, 21:1997-2002[Abstract]
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, BSA-AGE; 
, ovalbumin-AGE; •, casein-AGE; 
, dimer of methyl-glioxal-lysine (MOLD); 
, fructosyl-lysine, X, unglycated-BSA; ||, methyl glyoxal-ß-lactoglobulin. Early products of glycation, fructosyl lysine and unglycated BSA, showed no competitive activity. These data indicate that anti-AGE serum recognized glycated proteins and the developed competitive ELISA detected glycated proteins with sensitivity partially depending on the proteic substrate of glycation. Data represent means ± SD of three experiments performed in duplicate. Inset: Competitive ELISA calibration curve obtained by using glycated ß-lactoglobulin as reference. Within this molecule, an average of 4 mol of sugar (AGE) per mole of protein is present. This curve was then used to calculate the pmol of AGE per pmol of FGF2 under our conditions. The exact amount of AGE in ß-lactoglobulin was determined by electrospray mass spectrometry as described.
