Advanced glycation of apolipoprotein A-I impairs its anti-atherogenic properties
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AGE contribute to the pathogenesis of diabetic complications, including dyslipidaemia and atherosclerosis. However, the precise mechanisms remain to be established. In the present study, we examined whether AGE modification of apolipoprotein A-I (apoA-I) affects its functionality, thus altering its cardioprotective profile.
Materials and methods
The ability of AGE-modified apoA-I to facilitate cholesterol and phospholipid efflux, stabilise ATP-binding cassette transporter A1 (ABCA1) and inhibit expression of adhesion molecules in human macrophages and monocytes was studied.
The ability of AGE-modified apoA-I to promote cholesterol efflux from THP-1 macrophages, isolated human monocytes and from ABCA1-transfected HeLa cells was significantly reduced (>70%) compared with unmodified apoA-I. This effect was reversed by preventing AGE formation with aminoguanidine or reversing AGE modification using the cross-link breaker alagebrium chloride. AGE-modification of HDL also reduced its capacity to promote cholesterol efflux. AGE–apoA-I was also less effective than apoA-I in stabilising ABCA1 in THP-1 cells as well as in inhibiting expression of CD11b in human monocytes.
AGE modification of apoA-I considerably impairs its cardioprotective, antiatherogenic properties, including the ability to promote cholesterol efflux, stabilise ABCA1 and inhibit the expression of adhesion molecules. These findings provide a rationale for targeting AGE in the management of diabetic dyslipidaemia.
KeywordsAGE Atherosclerosis Diabetes High-density lipoprotein Inflammation Reverse cholesterol transport
ATP-binding cassette transporter A1
ATP-binding cassette transporter G1
Acetylated low density lipoprotein
Coronary artery disease
Human serum albumin
Liver X receptor
Phorbol myristate acetate
Reverse cholesterol transport
Both type 1 and type 2 diabetes are associated with increased risk of developing atherosclerosis and coronary artery disease (CAD) [1, 2]. While diabetic dyslipidaemia appears to be an important pro-atherogenic factor [3, 4], changes in the lipid profile are not sufficient to explain the increased cardiovascular risk observed in diabetes. Moreover, diabetes per se has both independent and additive contributions over and above that of dyslipidaemia towards the development and progression of atherosclerotic lesions [5, 6]. This suggests that not only quantitative, but also functional changes in lipid metabolism induced by diabetes may contribute significantly to the accelerated atherosclerosis associated with diabetes. Hence, not only plasma concentrations of lipoproteins, but also their functional properties are affected by glucose-induced modifications and may be critical for diabetes-induced atherosclerosis.
Functional changes in both forward and reverse cholesterol transport may be important in mediating diabetes-associated atherosclerosis. While the role of diabetes-induced changes in the forward cholesterol transport branch of lipoprotein metabolism has been thoroughly investigated , information on the effect of diabetes on reverse cholesterol transport (RCT) is limited. Type 2 diabetes is consistently associated with reduced levels of HDL , and the composition, and possibly the functionality, of HDL particles are also altered in diabetic patients . The rate-limiting step of RCT is cholesterol efflux, which contributes both to the regulation of plasma HDL levels  and to maintaining macrophage cholesterol homeostasis . Cholesterol efflux in diabetic patients can be impaired by a number of mechanisms, including the effects of increased concentration of fatty acids  or glucose  on ATP-binding cassette, transporter A1 (ABCA1). Further, AGE modification of ABCA1 inhibits its function as well as its abundance in the macrophages of diabetic patients . Another factor likely to contribute to the impairment of cholesterol efflux is the modification of HDL and apolipoprotein A-I (apoA-I) induced by factors associated with diabetes, such as nitration . Simple glycation of HDL apparently does not affect its ability to promote cholesterol efflux ; however, non-enzymatic glycosylation of HDL has been reported to impair its ability to bind to the cell surface receptors on human fibroblasts  and to support the efflux of intracellular cholesterol . The potential effects of advanced glycation of apoA-I and the majority of the elements of RCT have not been examined previously. In the present study, we assess for the first time the effect of AGE modification of HDL and apoA-I on RCT and other anti-atherogenic properties.
Materials and methods
THP-1 cells (a human monocyte–macrophage cell line) were cultured in RPMI 1640, 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l glutamine (all reagents were from JRH Biosciences, Brooklyn, VIC, Australia). Cells were seeded at a density of 106 cells per well in 12-well tissue culture plates and cultured for 48 h. Phorbol myristate acetate (PMA) was added to differentiate the cells at the final concentration of 100 ng/ml. When indicated, cells were treated for 18 h with the liver X receptor (LXR) agonist T0-901317 (Sigma, St Louis, MO, USA) at the final concentration of 1 μmol/l.
HeLa cells were transfected with ABCA1 or ATP-binding cassette transporter G1 (ABCG1) (a kind gift of A. Remaley) using Lipofectamine Plus Reagent (Invitrogen, Mount Waverley, VIC, Australia) according to the manufacturer’s recommendations. Cells were treated for 18 h with 1 μmol/l 5′-azacytidine to prevent methylation of the cytomegalovirus (CMV) promoter .
Resting human monocytes were isolated from whole blood using the Dynal negative isolation kit (Invitrogen) .
HDL was isolated by sequential centrifugation from plasma obtained from healthy volunteers (n = 5) and patients with diabetes and kidney disease (n = 10). Samples were provided with the written consent of all participants and the approval of the Austin Health human ethics committee. HDL for in vitro experiments was isolated from pooled plasma provided by the Red Cross. ApoA-I was isolated as described previously . Reconstituted HDL (rHDL) (apoA-I/POPC 1:80 mol/mol) was prepared as described previously .
Lipid-free apoA-I, isolated plasma HDL and human serum albumin (HSA) from healthy volunteers were modified by incubation with ribose (final concentration 0.5 mol/l) at 37°C for 18 h followed by dialysis with PBS, and further incubated for 5 days unless indicated otherwise. Glycated samples were dialysed extensively and sterilised by filtration.
To account for changes introduced by prolonged incubation, control preparations were also treated under identical conditions but without ribose (designated here as ‘treated’ proteins). Carboxymethyllysine (CML) levels were measured by indirect ELISA and expressed as μg CML/mg protein. Because of considerable variation in the AGE levels in human samples, results are expressed as their geometric mean. In vitro modification with methylglyoxal (MGO) has also been suggested to provide a good representation of physiological AGE modification [21, 22]. Purified apoA-I was modified by incubation with the indicated concentration of MGO at 37°C for 18 h. Excess MGO was removed by dialysis before samples were further incubated at 37°C for 72 h. Samples were then dialysed extensively and sterilised by filtration. In additional experiments, human apoA-I was also glycated by incubation with d-glucose (final concentration 0.5 mol/l) in the presence or absence of aminoguanidine (450 nmol/l) at 37°C for the indicated period of time.
Cholesterol and phospholipid efflux
Cellular cholesterol or phospholipids were labelled by incubation in serum-containing medium with [1α,2α(n)-3H]cholesterol (GE Healthcare-Amersham, Rydalmere, NSW, Australia; specific radioactivity 1.81 TBq/mmol, final radioactivity 0.5 MBq/ml) or [methyl-14C]choline (0.2 MBq/ml) for 48 h at 37°C in a CO2 incubator. After labelling, cells were washed and further incubated for 18 h in serum-free medium. Cells were then washed and incubated for 2 h at 37°C in serum-free medium containing either lipid-free apoA-I or rHDL (final concentration 30 μg/ml) or isolated HDL (final concentration 40 μg/ml). The medium was collected and centrifuged to remove cellular debris, and the radioactivity in the supernatant fractions was measured. Cells were harvested and dispersed in 0.5 ml distilled water, and aliquots were counted. For phospholipid efflux, phospholipids were isolated from medium and cells by thin-layer chromatography as described previously , and radioactivity was measured. Cholesterol and phospholipids efflux were expressed as the percentage of labelled cholesterol or phospholipid transferred from cells to the medium.
To assess cholesterol esterification, cells were incubated for 2 h at 37°C with [14C]oleic acid (GE Healthcare-Amersham; specific activity 2.22 GBq/mmol; final radioactivity 0.185 MBq/ml) complexed to BSA (Sigma; essentially fatty-acid-free). Where indicated, cells were preloaded with cholesterol by incubation for 24 h with acetylated LDL (AcLDL) (final concentration 50 μg/ml). Cells were washed and lipids were extracted and analysed by thin-layer chromatography as described previously . Spots of cholesterol and cholesteryl oleate were identified by standards (Sigma), scraped and counted in a beta-counter.
Cells were grown as described above and ABCA1 expression was induced by incubation for 18 h with the LXR agonist T0-901317 (1 μmol/l). Cells were then washed and incubated for 18 h at 37°C in the presence or absence of apoA-I or AGE-apoA-I (final concentration 30 μg/ml). Cells were then lysed by incubation in 5 mmol/l Tris–HCl (pH 7.5) containing protease inhibitor cocktail (Roche, Kew, VIC, Australia) at 4°C and soluble membrane protein fractions were isolated as described in . ABCA1 abundance was then analysed by western blot using monoclonal anti-ABCA1 antibody and quantitated using the Quantity One documentation system (Bio-Rad, Regents Park, NSW, Australia).
Expression of CD11b on human monocytes
Resting human monocytes were isolated from whole blood and resuspended at 106 cells/ml. One hundred microlitres of the suspension was stimulated with PMA (final concentration 100 ng/ml) in the presence or absence of apoA-I, treated apoA-I or AGE-apoA-I (40 μg/ml) and incubated with fluorescein isothiocyanate (FITC)-conjugated antibody against the active epitope of CD11b for 15 min at 37°C. Cells were then fixed with 4% paraformaldehyde. Controls used were unstimulated monocytes and the isotype-matched irrelevant antibody (FITC-anti-mouse IgG). CD11b expression was measured as fluorescence intensity by the use of a FACSCalibur flow cytometer (Beckman, Glagesville, NSW, Australia). Analysis was conducted using the Cell Quest Pro software (BD Biosciences, San Jose, CA, USA).
THP-1 cells were differentiated and cultured for 72 h on sterile collagen-coated glass coverslips. Cells were washed with PBS then incubated with apoA-I or AGE-apoA-I in serum-free media for 18 h at 37°C, washed with PBS and fixed in acetone for 10 min at –20°C. This was followed by incubation for 1 h with purified monoclonal anti-ABCA1 antibody (5 μg/ml), and with secondary Alexa Fluor anti-mouse IgM antibodies (5 μg/ml). Cells were observed using a Zeiss Meta confocal microscope.
All experiments were replicated two to four times and representative experiments are shown unless indicated otherwise. Means±SD of quadruplicate determinations are shown. Student’s t test was used to determine the statistical significance of the differences. Correlation was analysed using Spearman rank order correlation.
AGE modification of apoA-I and HDL
To assess the possible level of AGE modification of HDL in diabetic patients, HDL was isolated from plasmas of ten diabetic subjects with kidney disease (a group of patients known to have high levels of circulating AGE); CML levels in plasma protein and isolated HDL were compared with those of five healthy individuals. The levels of CML in patient plasmas were 1.7-fold higher than those in the plasmas of healthy subjects (3.0 ± 1.7 vs 1.8 ± 0.5 μg/mg protein). By contrast, the concentration of CML in HDL fractions was almost tenfold higher in diabetic subjects with kidney disease compared with healthy subjects and similar to the levels of CML observed in apoA-I and HDL modified in vitro (Fig. 1c).
Cholesterol and phospholipid efflux to AGE-modified apoA-I
To investigate whether impairment of cholesterol efflux to AGE-modified apoA-I affects the ABCA1-dependent pathway, ABCA1 expression in the cells was boosted by overnight treatment with the LXR agonist T0-901317. This treatment significantly increased the rate of cholesterol efflux. However, AGE modification of apoA-I with ribose reduced LXR agonist-induced cholesterol efflux by 70% (Fig. 2a). ApoA-I incubated without ribose retained had an ability to support cholesterol efflux from activated cells similar to that of untreated apoA-I (Fig. 2a). To further confirm the involvement of the ABCA1-dependent cholesterol efflux pathway, the effect of AGE modification of apoA-I on phospholipid efflux was tested. The efflux of phospholipid to AGE-apoA-I from THP-1 cells activated with T0-901317 was halved compared with the efflux to unmodified apoA-I (Fig. 2c).
To exclude the possibility of a non-specific effect of AGE moiety on cholesterol efflux, THP-1 cells were pre-incubated for 18 h with AGE-modified HSA. No effect of AGE-modified HSA on cholesterol efflux was observed when HSA was either removed from the incubation mixture before adding apoA-I or when AGE HSA was left in the incubation mixture during the efflux (3.9 ± 0.5 and 3.8 ± 0.2% for unmodified and AGE-modified HSA respectively).
Effects of inhibitors of advanced glycation on cholesterol efflux
The effect of AGE modification of apoA-I on cholesterol efflux was further tested using two compounds that attenuate AGE modification. First, we used the putative AGE crosslink breaker alagebrium chloride (ALT-711) . When AGE-modified apoA-I was pretreated with 0.1 mg/ml alagebrium chloride, its ability to support cholesterol efflux was fully restored (Fig. 2d). Second, we used aminoguanidine, which prevents the formation of AGE . Prolonged incubation of apoA-I with aminoguanidine enhanced the ability of apoA-I to promote cholesterol efflux; however, cholesterol efflux was similar for apoA-I and apoA-I modified in the presence of aminoguanidine (Fig. 2d).
Alternative pathways for AGE modification of apoA-I
Another physiologically relevant mechanism of AGE modification is through the action of MGO. MGO levels are elevated in diabetes [21, 22], in which they correlate with the extent of AGE modification better than glucose levels. In our experiments, various concentrations of MGO sharply reduced the functionality of apoA-I towards cholesterol efflux (Fig. 3c).
Cholesterol efflux to AGE-modified HDL
Most of the apoA-I in plasma is lipidated and is a constituent of HDL. Therefore the effect of the AGE modification of HDL on its ability to promote cholesterol efflux was also examined. Cholesterol efflux to HDL is likely to be mediated by several pathways, including ABCG1- and SR-B1-dependent pathways. When cholesterol efflux from THP-1 cells to HDL was tested, there was a statistically significant reduction of cholesterol efflux to AGE-modified HDL, whereas the efflux to HDL incubated in the absence of ribose was similar to that in untreated samples (Fig. 3d).
Mechanisms of impairment of cholesterol efflux to AGE-modified apoA-I and HDL
Intracellular cholesterol content
AGE modification of apoA-I and ABCA1 stability
AGE modification of apoA-I and expression of CD11b on monocytes
The presence of high levels of AGE in plasma is a characteristic feature of diabetes, with mounting evidence of a significant role of AGE in diabetic complications . High plasma concentrations of AGE have been associated with increased rates of CAD  as well as kidney damage . AGE modifications have also been reported to cause changes in lipoprotein metabolism affecting properties of LDL particles , expression of lipoprotein lipase  and the functionality of scavenger receptors, including SR-B1 . Most of these effects were related to common AGE moieties, such as CML , and not related to a specific protein. In addition to the effects of AGE moiety, AGE modification can affect specific functions of specific proteins contributing to the impairment of metabolic regulation. For example, AGE modification of ABCA1 has a dramatic effect on cholesterol efflux . Persistent hyperglycaemia and oxidative stress in diabetes act to hasten the formation of AGE, ensuring not only that long-lived proteins become more heavily modified, but also that short-lived molecules such as apoA-I become targets for AGE modification . In addition to their role in RCT, apoA-I and HDL have other anti-atherogenic functions, including anti-inflammatory , antioxidant  and antithrombotic actions . Hence, AGE modification of apoA-I may have wide-ranging implications for the development of atherosclerosis, particularly in the setting of diabetes. In this study we investigated the effect of AGE modification of apoA-I on several of its anti-atherogenic functions.
The main finding of this study is that a number of the known anti-atherogenic functions of apoA-I were impaired by a physiologically relevant level of AGE modification of this specific protein. These functions include cholesterol efflux. The cholesterol efflux pathway affected was likely to be mainly, but not exclusively, the ABCA1-dependent pathway. This conclusion is supported by the greater inhibition of cholesterol efflux to AGE-apoA-I compared with AGE–HDL, concomitant inhibition of phospholipid efflux and almost complete inhibition of cholesterol efflux to AGE-apoA-I from ABCA1-transfected HeLa cells, where ABCA1-dependent cholesterol efflux is the only pathway for removing cholesterol to apoA-I. However, inhibition of cholesterol efflux to AGE–HDL indicates that the other cholesterol efflux pathways, including the ABCG1-dependent pathway, are also affected. The changes in cholesterol efflux were specific for AGE-modification of apoA-I as AGE-HSA had no effect on cholesterol efflux. The reduced ability of AGE-apoA-I to support cholesterol efflux was reflected in its reduced capacity to decrease cell cholesterol content as assessed by the rate of cholesterol esterification. The impairment of cholesterol efflux was reversed by preventing or reversing AGE modification of apoA-I. Interestingly, simple glycation of apoA-I does not seem to affect its ability to promote cholesterol efflux , emphasising that further modification with the formation of AGE is necessary for impairment of apoA-I function. Our finding is consistent with that of Brubaker et al.  showing that reductive methylation of lysine residues impairs the ability of apoA-I to support cholesterol efflux, and with that of Duell et al.  showing that non-enzymatic glycosylation of HDL impairs its function. Thus, AGE-modification of apoA-I and HDL results in a significant loss of its ability to support cholesterol efflux, which can be reversed by either inhibiting or reversing AGE-modification. These findings provide a rationale for targeting AGE as part of the management of diabetic dyslipidaemia and cardioprotection.
Other functions of apoA-I impaired by AGE-modification included its ability to stabilise ABCA1  and to inhibit expression of adhesion molecules . The former further impairs RCT, and the latter is a reflection of the anti-inflammatory function of apoA-I. Inflammation is an important element in the pathogenesis of atherosclerosis and the anti-inflammatory properties of apoA-I are likely to contribute to its overall anti-atherosclerotic effect .
The clinical relevance of AGE modification to the functions of apoA-I in vivo remains to be tested. Although the concentration of CML found in the in vitro-modified apoA-I used in this study was similar to that found in vivo, it is conceivable that other AGE-modifications may contribute to protein dysfunction, and may be different from that observed in vivo. Moreover, AGE modification is not the only apoA-I modification observed in diabetes that potentially contributes to impaired apoA-I functions. In particular, nitration and chlorination of apoA-I also occur in diabetes, damaging apoA-I functionality [13, 45, 46]. While the relative contribution of AGE modification to the overall effect of diabetes on apoA-I dysfunction is yet to be established, it is clear that interventions to reduce AGE levels in diabetes are anti-atherosclerotic .
In conclusion, we demonstrated that AGE modification of apoA-I and HDL results in a significant loss of their ability to support cholesterol efflux, stabilise ABCA1 and inhibit the expression of CD11b. These changes appear to be specific to the AGE modification of apoA-I and can be attenuated by preventing AGE modification. The impaired functioning of AGE-modified apoA-I may contribute significantly to the increased risk of atherosclerosis associated with diabetes.
We are grateful to A. Remaley for the ABCA1 and ABCG1 plasmids. This study was supported by a grant from the Diabetes Australia Research Trust (D. Sviridov, R. O’Brien) and from the Juvenile Diabetes Research Foundation (J. M. Forbes, M. T. Coughlan, M. C. Thomas). D. Sviridov and J. P. F. Chin-Dusting are fellows of the National Health and Medical Research Council of Australia.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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