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Elevated serum levels of AGEs, sRAGE, and pentosidine in Tunisian patients with severity of diabetic retinopathy
Microvascular Research 84 (2012) 378–383
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Elevated serum levels of AGEs, sRAGE, and pentosidine in Tunisian patients with severity of diabetic retinopathy Mohsen Kerkeni a,⁎, Amel Saïdi a, Hassan Bouzidi b, Slim Ben Yahya c, Mohamed Hammami a a b c
Laboratory of Biochemistry, UR: Human Nutrition & Metabolic Disorders, Faculty of Medicine, Monastir University, Tunisia Biochemistry Department, CHU Tahar Sfar, Mahdia, Tunisia Ophthalmology Department, CHU Fattouma Bouguiba, Monastir, Tunisia
a r t i c l e
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Article history: Accepted 17 July 2012 Available online 24 July 2012
a b s t r a c t Objectives: The advanced glycation end products (AGEs)-receptor for AGE (RAGE) axis activity are implicated in diabetic vascular complications. We measured serum AGE, sRAGE and pentosidine levels in Tunisian patients with diabetic retinopathy (DR) and examined whether these biomarkers are related to the severity of DR. Design and methods: We included 30 healthy control subjects and 100 diabetic patients were divided into 2 subgroups: 40 patients with nonproliferative diabetic retinopathy (NPDR), and 60 patients with proliferative diabetic retinopathy (PRD). AGEs, sRAGE and pentosidine were measured in serum by ELISA. Results: Serum levels of AGEs, sRAGE and pentosidine were significantly increased in patients with diabetes mellitus compared to nondiabetic controls (P b .01, P b .001, P b .001 respectively). In diabetic patients, serum AGEs, sRAGE and pentosidine levels were significantly higher in patients who had PDR than in those with NPDR (P = .001, P = .01, P = .005 respectively). Furthermore, in stepwise multivariate regression analysis, the levels of pentosidine and duration of diabetes were independently associated with severity of DR. Conclusion: Serum AGEs, sRAGE, and pentosidine levels are related with the presence of DR. Duration of diabetes and pentosidine were independently correlated with the severity of DR. © 2012 Elsevier Inc. All rights reserved.
Introduction Diabetes mellitus (DM) is increasing at an alarming rate in Tunisian population; patients are prone to the development of macroand microvascular diabetic complications that represent a major cause of morbidity and mortality. DM is now a global pathology, with recent surveys predicting that by 2025, the number of patients with diabetes will rise to staggering 380 million (King et al., 1998; WHO, 2011). This worldwide disease will lead to increasing incidence of the two major types of late complications: macrovasuclar and microvascular that contribute to morbidity and premature deaths. The macrovascular complications, which affect the large vessels include cardiovascular, cerebrovascular, and peripheral vascular disease. The microvascular complications, which affect the small vessels, involve neuropathy, nephropathy, and retinopathy. Diabetic retinopathy (DR) is one of the most significant complications of DM,
Abbreviations: AGEs, Advanced glycation end products; DM, Diabetes mellitus; DR, Diabetic retinopathy; NPDR, Nonproliferative diabetic retinopathy; PRD, Proliferative diabetic retinopathy; sRAGE, soluble form of receptor for advanced glycation end products. ⁎ Corresponding author. E-mail address: [email protected] (M. Kerkeni). 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.mvr.2012.07.006
it occurs in 90% of patients after 20–30 years from the disease diagnosis, and its advanced form, proliferative diabetic retinopathy (PDR), affects over 60% of diabetic patients (Fong et al., 2003). The major risk factors for DR are hyperglycemia, high blood pressure, and duration of diabetes. Chronic hyperglycemia is a major vascular complication of DM. Research worldwide has established various biochemical mechanisms that potentially link hyperglycemia and DR. These pathogenic mechanisms include polyol pathway flux, activation of diacylglycerol (DAG)-PKC pathway, increased expression of growth factors, accelerated formation of advanced glycation end products (AGEs), oxidative stress, hemodynamic or retinal blood flow changes, rennin–angiotensin system activation, and sub-clinical inflammation (Tarr et al., 2011). Recently, AGEs have become more and more focused in this issue. These are formed by the Maillard process, a non-enzymatic reaction between ketones or aldehydes, the amino groups of proteins, lipids and nucleic acids that contributes to the aging of macromolecules (Dyer et al., 1991; Stern et al., 2002; Takeuchi and Yamagishi, 2009). Under hyperglycemic and/or oxidative stress conditions, this process begins with the conversion of reversible Schiff base adducts to more stable, covalently-bound Amadori rearrangement products (Dyer et al., 1991). Over the course of days to weeks, these Amadori products undergo further rearrangement reactions to form the irreversibly-bound moieties known as AGEs. Among
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the few advanced glycation products characterized to date, pentosidine is chemically well defined (Odetti et al., 1992). It is synthesized through nonenzymatic reactions of pentose, and its formation is closely related to oxidative processes (Miyata et al., 1998). Plasma pentosidine was reported to be significantly higher in patients with diabetes than in subjects without diabetes and associated with in an increased incidence of cardiovascular disease and increased stiffening and thickening of the large arteries in patients with type 2 diabetes (Yoshida et al., 2005). Previous studies have confirmed that AGEs and receptor for AGEs (RAGE) interaction elicits oxidative stress generation in various types of cells and subsequently evokes vascular inflammation, macrophage and platelet activation, and thrombosis, thereby playing an important role in the development and progression of vascular complications in diabetes (Bierhaus et al., 1998; Takenaka et al., 2006; Yamagishi et al., 2008a,b). Several different receptors for AGEs have been identified, one of which is termed RAGE. Different cell types including human endothelial cells express RAGE (Yonekura et al., 2003). In addition to cell-bound RAGE, soluble forms of RAGE appear in the plasma, as different splice variants of RAGE (e.g. endogenous secretory receptor for AGE, esRAGE) leaking through the transmembrane and cytosolic domain, and as a proteolytically cleaved form of RAGE (sRAGE), which is most probably shed into the circulation by the sheddase, known as a disintegrin and metalloprotease 10 (ADAM 10) (Hudson et al., 2008; Raucci et al., 2008; Schlueter et al., 2003). The functional role of these soluble forms of RAGE in the circulation remains unclear, but they may reflect the activity of the AGE-RAGE axis. The ligation of RAGE activates the endothelial cell and triggers multiple signaling cascades resulting in activation and translocation of nuclear transcription factors and transcription of the target genes, including adhesion molecules and proinflammatory cytokines (Basta et al., 2002; Harja et al., 2008; Hofmann et al., 1999; Kislinger et al., 1999). Biological function of sRAGE has not been clearly defined and its biological importance within the AGE-RAGE axis is only recently beginning to be understood. In view of these considerations, we have investigated whether AGEs, sRAGE and pentosidine are associated with the presence and the severity of DR; to date little reports have examined the relationship between these three biomarkers and the presence and severity of DR. In the present study, we measured serum AGE, sRAGE and pentosidine levels in Tunisian patients with DR and examined whether these biomarkers are related to the severity of DR. Subjects and methods Subjects The local ethics committee approved this study. Written informed consent was obtained from all patients before the enrollment. In this prospective cohort study, we measured serum concentration of AGEs, sRAGE and pentosidine in 130 participants (age range, 50–75 years). All patients were diagnosed by ophthalmoscopy and fundus stereophotography after dilatation by specialized ophthalmologist in University Hospital Fattouma Bourguiba at Monastir (Tunisia). The severity of the retinopathy was categorized as nonproliferative DR (NPDR, mildto-severe NPDR), or proliferative DR (PDR, level >60). The subjects were divided into 2 groups: Group I is composed of healthy volunteers (n = 30) with no DM or systemic or local eye lesion. Group II is composed of DM patients (n = 100) who developed DR. This group showed DM patient who developed NPDR (n = 40) and DM patient who developed PDR (n = 60). For each patient, a data sheet was completed with the patient's identification code, age, sex, and duration of diabetes, and the antidiabetic agent used, if any. For cardiovascular risk factors, the following definitions were used: individuals were defined as hypertensive if their blood pressure was > 140/90 mm Hg or if they were receiving any antihypertensive treatment; individuals were deemed dyslipidemic when their total cholesterol concentration was
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≥ 5.68 mmol/l, or their triglyceride concentration was ≥2.28 mmol/l, or they receiving lipid-lowering drugs. Methods In all subjects, venous blood was collected in the morning after an overnight fast. The samples were stored at − 80 °C until analysis. Random plasma glucose, hemoglobin A1C (HbA1C) was measured using G7 HPLC Analyser (Tososh Europe N.V), serum creatinine, uric acid, and lipid levels (HDL, LDL, cholesterol, and triglyceride) were measured using enzymatic methods by CX9 Auto-chemical analysis instrument (Beckman CX9, USA). AGEs, sRAGE, and pentosidine were quantitatively determined in serum by enzyme-linked immunosorbent assay (ELISA) kits provided by ABO Switzerland Co., Ltd, according to the manufacturer's instructions. Briefly, the microtiter plate has been pre-coated with an antibody specific to Human AGEs (or sRAGE or Pentosidine). Samples were added, after incubation and washing, plates were incubated with HRP, developed with TMB substrate, and OD450 was determined using an ELISA plate reader. Statistical analysis All values are expressed as mean ± SD. A P value less than 0.05 was considered statistically significant. Significance between 2 groups was determined by independent sample Student t-test for continuous variables. Continuous data from > 2 groups were compared with 1-way analysis of variance (ANOVA). All analyses were performed using the SPSS program (version 17). Correlation was determined by linear regression analysis. Multiple regression was used to further explore the linear relationships between the variables. The equation was in the form: y =a + b1 × x1+ b2 × x2 + … + bp × xp. Regression variables were estimated as well as the correlation coefficient r. ANOVA was used to assess the significance of the regression with significance accepted at P b 0.05. As an alternative data evaluation method, stepwise backward regression analysis was also used on the variables. Results General clinical and biochemical parameters of the studied groups The clinical characteristics and laboratory data of controls and diabetic patients are shown in Table 1. Significant differences were seen between the groups in body mass index, HbA1C, serum glucose, lipids profiles and uric acid but not in serum creatinine. Serum levels of AGEs, sRAGE and pentosidine were significantly increased in diabetic patients than in controls (667.98± 198.51 vs. 508.83 ± 119.68 pg/mL; 206.45 ± 53.18 vs. 148.72 ±32.73 pg/mL; 337.79 ± 63.16 vs. 246.03 ± 55.05 pg/mL, P b 0.01; P b 0.001; and P b 0.001 respectively). Relationship between AGEs, sRAGE, pentosidine levels and severity of DR Table 2 show results of serum AGEs, sRAGE, pentosidine levels in patients with NPDR and patients with PDR and illustrated in Figs. 1, 2 and 3. Patients with PDR showed an increased level of serum AGEs, sRAGE, and pentosidine than patients with NPDR (751.48 ± 209.52 vs. 533.54 ± 82.92 pg/mL; 223.21 ± 62.57 vs. 178.54 ± 11.16 pg/mL; 361.70 ± 66.60 vs. 300.33 ± 36.49 pg/mL, P = 0.001, P = 0.01 and P = 0.005 respectively). Duration of diabetes was higher in patients with PDR than patients with NPDR (P = 0.001). Linear regression and multivariate analyses By linear regression analysis in the 100 diabetic patients with retinopathy, both AGEs and pentosidine levels correlated positively with age, duration of diabetes and serum creatinine (Table 3). sRAGE levels correlated positively with AGEs and creatinine. Furthermore, serum
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Table 1 Clinical characteristics of control subjects and diabetic patients.
N (M/F) Age (years) BMI (kg/m2) Duration of diabetes (years) Hypertension, n (%) Dyslipidemia, n (%) Glucose (mmol/L) HbA1C (%) Total cholesterol (mmol/L) Triglyceride (mmol/L) HDL (mmol/L) LDL (mmol/L) Creatinine (μmol/L) Uric acid (μmol/L) AGEs (pg/mL) sRAGE (pg/mL) Pentosidine (pg/mL)
Control subjects
Diabetic patients
P
30 (17/13) 52 ± 9 25.6 ± 1.2 – 0 (0) 0 (0) 4.77 ± 0.91 5.6 ± 0.2 4.55 ± 0.74 1.42 ± 0.68 1.52 ± 0.47 2.25 ± 0.45 88. 91 ± 15.53 209.06 ± 47.54 508.83 ± 119.68 148.72 ± 32.73 246.03 ± 55.05
100 (53/47) 59 ± 11 29.4 ± 2.4 14.93 ± 8.03 74 (74) 36 (36) 12.35 ± 3.94 8.14 ± 1.6 5.22 ± 0.12 2.15 ± 0.98 1.20 ± 0.31 2.77 ± 0.33 102.42 ± 20.33 287.91 ± 52.80 667.98 ± 198.51 206.45 ± 53.18 337.79 ± 63.16
– NS b 0.05 – b 0.001 b 0.001 b 0.001 b 0.001 b 0.01 b 0.05 b 0.05 b 0.05 NS b 0.01 b 0.01 b 0.001 b 0.001
Values are mean ± SD. BMI: body mass index; NS: not significant.
pentosidine correlated positively with total cholesterol and uric acid. Multiple linear regression was used in patients with PDR to identify any related predictors of severity of DR, evaluating the regression coefficients that represent the contributions of each independent variable to the predictive value of the dependent variable (PDR) (Table 4). This model, which was applied to duration of diabetes, AGEs, sRAGE and pentosidine variables (considered as independent variables), as well as PDR as the dependent variable, showed that duration of diabetes and pentosidine correlated positively.
Discussion Predictors of diabetic complications may be important in the prevention and the management of these complications (van Leiden et al., 2003). The present data demonstrated that duration of diabetes, AGE, sRAGE, and pentosidine levels are positively associated with the presence of DR and duration of diabetes and pentosidine were independently related to PDR. DR is a duration-dependent disease that develops in stages, the incidence of retinopathy is rarely detected in the first few years of diabetes, but the incidence increases to 50% by 10 years and to 90% by 25 years of diabetes (Kowluru and Chan, 2007). As duration of diabetes is a total reflection of blood glucose control and exposing to other risk factors, and the prevalence of DR is highly related with the increasing of HbA1c, duration of diabetes is an important factor for the incidence and development of DR. Similar conclusions were also found in several studies which demonstrated that duration of diabetes and hyperglycemia contributed to the development of DR, and if excellent glycemic control was done at the beginning, DR can be controlled (Herman et al., 1998; McKay et al., 2000). During diabetes, the rate of formation of AGEs exceeds that by first-order kinetics. Thus, over time, even modest hyperglycemic
Fig. 1. Box plots of serum AGEs levels in patients with NPDR and PDR. The horizontal lines in each box plot represent (bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles.
excursions can result in significant adduct accumulation on long-lived macromolecules (Monnier et al., 2005). A number of clinical studies have reported that the formation of AGEs and accumulation have been found in retinal blood vessels of diabetic patients and animals and in human serum and vitreous of diabetic patients which were found to correlate with the degree of DR (Goh and Cooper, 2008). Retinal pericytes which play an important role in the maintenance of microvascular homeostasis have been shown to accumulate AGEs during diabetes which are implicated in endothelial cell injury and blood-retinal barrier dysfunction (Stitt et al., 2000). In addition, AGEs increases vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) expression in microvascular endothelial cells through incellular ROS generation (Yamagishi et al., 2007a). AGEs also activate nuclear factor-B (NF-κB) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase with increase in ROS and apoptosis of pericytes and other retinal cells (Ibrahim et al., 2011; Yamagishi and Matsui, 2011). AGEs disturb microvascular homeostasis through interaction with RAGE. This AGE-RAGE axis plays a central role in the inflammation, neurodegeneration, and microvascular dysfunction in DR (Yamagishi, 2009). A previous study reported that among AGEs in patients with type 2 diabetes, high serum pentosidine is associated with both increased
Table 2 AGEs, sRAGE, and pentosidine levels in diabetic patients with and without PDR patients with DR. Patients with DR
Duration of diabetes (years) HbA1C (%) AGEs (pg/mL) sRAGE (pg/mL) Pentosidine (pg/mL) Values are mean ± SD.
NPDR (n = 40)
PDR (n = 60)
P
8.70 ± 3.16 7.84 ± 1.2 533.54 ± 82.92 178.54 ± 11.16 300.33 ± 36.49
18.29 ± 8.10 8.86 ± 1.5 751.48 ± 209.56 223.21 ± 62.57 361.70 ± 66.60
0.001 0.015 0.001 0.010 0.005
Fig. 2. Box plots of serum sRAGE levels in patients with NPDR and PDR. The horizontal lines in each box plot represent (bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles. Values above the 90th are plotted as points.
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Table 4 Variables estimates for multiple linear regression applied to AGEs, sRAGE, pentosidine and duration of diabetes in diabetic patients with PDR. Term
Estimate
Standard error
t Ratio
P
Intercept AGEs sRAGE Pentosidine Duration of diabetes
56.867 −0.256 −0.048 4.340 21.340
28.336 0.162 0.019 1.182 5.282
2.87 −1.34 −1.37 3.31 3.58
0.034 NS NS 0.0014 0.0010
Co-linearity statistics Tolerance
VIF
0.224 0.243 0.936 0.631
4.628 4.154 1.598 1.098
PDR was the dependent variable; ANOVA revealed a statistically significant fit (P b 0.001) r = 0.755, r2 = 0.570, r2 adjusted = 0.457. VIF: variance inflation factor.
Fig. 3. Box plots of serum Pentosidine levels in patients with NPDR and PDR. The horizontal lines in each box plot represent (bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles. Values below the 10th percentiles are plotted as points.
carotid intima-media wall thickness and arterial stiffening (Yoshida et al., 2005). Recently, Sato et al. reported that plasma pentosidine levels increased significantly in patients with NPDR compared to patients with nondiabetic retinopathy. Authors found that retinal hemodynamic was correlated positively with plasma pentosidine (Sato et al., 2012). Furthermore, Sun et al. recently reported that specific AGE combinations (carboxyethyl-lysine and pentosidine) were strongly associated with complications indicating a link between AGE formation or processing with development of DR (Sun et al., 2011). For pentosidine, as a type of AGE, the study of Salman et al. was done to evaluate the role of pentosidine in predicting the progression of DR. The authors showed that significant elevation of pentosidine was found in patients during the earliest detectable phase of DR (early nonproliferative diabetic retinopathy) and more elevation at the preproliferative stage of retinopathy, returning to lower levels at the proliferative stage of diabetic retinopathy (Salman et al., 2009). However, our results showed that pentosidine levels were increased in diabetic patients with proliferative stage of DR. Indeed, the novelty of this study showed that pentosidine is also a biomarker for severity of DR. Ours current finding seems to agree with the results of a previous study but with small sample size (Ghanem et al., 2011). The present study demonstrated that by multivariate analysis, duration of diabetes and pentosidine was an independent factor increasing the grade of DR. This may reflect declining glomerular filtration rates in association both with aging and duration of diabetes because renal insufficiency may be a major determinant of serum pentosidine. We also found a positive correlation between serum pentosidine and serum creatinine or serum uric acid in diabetic
Table 3 Linear regression analysis of relationships between AGEs, sRAGE, pentosidine levels and characteristics of diabetic patients with retinopathy. Variables
Age (years) BMI Duration of diabetes HbA1C (%) AGEs (pg/mL) Total cholesterol (mmol/L) Triglyceride (mmol/L) Creatinine (μmol/L) Uric acid (μmol/L)
AGEs
sRAGE
Pentosidine
r
P
r
P
r
P
0.321 0.171 0.276 0.133 – 0.122 0.141 0.359 0.121
0.029 NS 0.031 NS – NS NS 0.022 NS
0.152 0.142 0.162 0.052 0.453 0.142 0.126 0.253 0.112
NS NS NS NS 0.008 NS NS 0.039 NS
0.443 0.143 0.353 0.125 0.321 0.243 0.153 0.273 0.253
0.014 NS 0.026 NS 0.029 0.041 NS 0.033 0.039
patients. Because the kidney is the main elimination site for pentosidine (Aso et al., 2004), several studies have examined serum pentosidine concentrations in patients with diabetes with overt nephropathy or those with chronic renal disease, reporting elevations (Miyata et al., 1996; Sugiyama et al., 1998; Suliman et al., 2003). Especially, a dramatic increase in plasma pentosidine was reportedly found in patients with end-stage renal disease (Weiss et al., 2000). Renal insufficiency thus may lead to accumulation of pentosidine in the blood because renal is the major determinant of serum pentosidine (Hricik et al., 1993). Formation of pentosidine requires oxidation as well as glycation. It might promote oxidative stress and endothelial cell dysfunction and induce the growth and tube formation of microvascular endothelial cells through autocrine vascular endothelial growth factor. We speculated that AGEs and pentosidine might accelerate the development and severity of DR both by accumulation of AGEs in the vessel walls and by causing endothelial dysfunction, as mediated by the AGE-RAGE axis activity. Moreover, we speculated that increased pentosidine might be involved in the stiffening of the retinal arteries. The increased levels of sRAGE across stage of severity of retinopathy observed in our study were also in line with following observations: (a) circulating sRAGE levels are increased, rather than decreased, in both type 1 and type 2 diabetic patients (Challier et al., 2005; Tan et al., 2006); (b) serum sRAGE levels are positively, but not inversely, correlated with circulating AGE levels in both non-diabetic and type 2 diabetic subjects (Tan et al., 2006; Yamagishi et al., 2006); (c) AGEs are positive regulators of cell surface expression of RAGE (Tanaka et al., 2000; Yamagishi and Takeuchi, 2004; Yamagishi et al., 2007b) and that RAGE is up-regulated in atherosclerostic plaques in diabetes, diabetic nephropathy and retinopathy (Cipollone et al., 2003; Pachydaki et al., 2006; Tanji et al., 2000); (d) vitreous levels of sRAGE are increased in proliferative retinal diseases by reflecting enhanced RAGE expression in epiretinal membranes of the eyes (Pachydaki et al., 2006). Treatment for reducing AGEs also prevents the onset or progression of DR. Because the current study had a small sample size, another clinical study of correlation between these biomarkers and the severity of DR is needed. In summary, the current study showed that serum AGEs, sRAGE and pentosidine levels increased in Tunisian patients with DR and duration of diabetes and pentosidine was independently correlated with severity of DR. Our results suggest that increased AGE-RAGE activity and increased of pentosidine may initiate and extent the severity of diabetic retinopathy. Intervention on serum AGEs, sRAGE and pentosidine may be a potential therapy for diabetes and its complications.
Conflict of interest statement The authors declare that they have no conflict of interest.
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Author contributions MK conceived of the study, carried out and performed the immunoassays for AGEs, sRAGE, pentosidine and analyzed statistical data and drafted the manuscript. AS recruited participants and prepared patients material for analysis. HB participated in routinely biochemical Laboratory measurements. SBY recruited patients and clinical data. MH conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgments This research was supported by a grant from the “Ministère de l'Enseignement Supérieur et de la Recherche Scientifique” UR03ES08 “Nutrition Humaine et Désordres Métaboliques” University of Monastir. We thank Prof. Mohsen Hassin and his colleagues of the regional blood transfusion centre in Monastir (Tunisia) for recruiting the healthy controls. Author Kerkeni express his sincere appreciation to Prof. Francois Trivin (Saint-Joseph Clinical Laboratory Medicine, Paris, France) for his continued direction and encouragement throughout the study. References Aso, Y., Takanashi, K., Sekine, K., Yoshida, N., Takebayashi, K., Yoshihara, K., et al., 2004. Dissociation between urinary pyrraline and pentosidine concentrations in diabetic patients with advanced nephropathy. J. Lab. Clin. Med. 144, 92–99. Basta, G., Lazzerini, G., Massaro, M., Simoncini, T., Tanganelli, P., Fu, C., et al., 2002. Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation 105, 816–822. Bierhaus, A., Hofmann, M.A., Ziegler, R., Nawroth, P.P., 1998. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. The AGE concept. Cardiovasc. Res. 37, 586–600. Challier, M., Jacqueminet, S., Benabdesselam, O., Grimaldi, A., Beaudeux, J.L., 2005. Increased serum concentrations of soluble receptor for advanced glycation endproducts in patients with type 1 diabetes. Clin. Chem. 51, 1749–1750. Cipollone, F., Iezzi, A., Fazia, M., Zucchelli, M., Pini, B., Cuccurullo, et al., 2003. The receptor RAGE as a progression factor amplifying arachidonate-dependent inflammatory and proteolytic response in human atherosclerotic plaques: role of glycemic control. Circulation 108, 1070–1077. Dyer, D.G., Blackledge, J.A., Thorpe, S.R., Baynes, J.W., 1991. Formation of pentosidine during nonenzymatic browning of proteins by glucose. Identification of glucose and others carbohydrates as possible precursors of pentosidine in vivo. J. Biol. Chem. 266, 11654–11660. Fong, D.S., Aiello, L., Gardner, T.W., 2003. American Diabetes Association, Diabetes retinopathy. Diabetes Care 26, 226–229. Ghanem, A.A., Elewa, A., Arafa, L.F., 2011. Pentosidine, and N-carboxymethyl-lysine: biomarkers for type 2 diabetic retinopathy. Eur. J. Ophthalmol. 21, 48–54. Goh, S.Y., Cooper, M.E., 2008. Clinical review: the role of advanced glycation end products in progression and complications of diabetes. J. Clin. Endocrinol. Metab. 93, 1143–1152. Harja, E., Bu, D.X., Hudson, B.I., Chang, J.S., Shen, X., Hallam, K., et al., 2008. Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/mice. J. Clin. Invest. 118, 183–194. Herman, W.H., Aubert, R.E., Engelgau, M.M., Thompson, T.J., et al., 1998. Diabetes mellitus in Egypt: glycaemic control and microvascular and neuropathic complications. Diabet. Med. 15, 1045–1051. Hofmann, M.A., Drury, S., Fu, C., Qu, W., Taguchi, A., Lu, Y., et al., 1999. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97, 889–901. Hricik, D.E., Schulak, J.A., Sell, D.R., Fogarty, J.F., Monnier, V.M., 1993. Effects of kidney or kidney–pancreas transplantation on plasma pentosidine. Kidney Int. 43, 398–403. Hudson, B.I., Cater, A.M., Harja, E., Kalea, A.Z., Arriero, M., Yang, H., et al., 2008. Identification, classification, and expression of RAGE gene splice variants. FASEB J. 22, 1572–1580. Ibrahim, A.S., El-Remessy, A.B., Matragoon, S., Zhang, W., Patel, Y., Khan, S., Al-Gayyer, M.M., El-Shishtway, M.M., Liou, G.I., 2011. Retinal microglial activation and inflammation induced by amadori-glycation albumin in a rat model of diabetes. Diabetes 60, 1122–1133. King, H., Aubert, R.E., Herman, W.H., 1998. Global burden of diabetes, 1995-2025: prevalence, numerical estimates, and projections. Diabetes Care 2, 1414–1431. Kislinger, T., Fu, C., Huber, B., Taguchi, A., Du Yan, S., Hofmann, M., et al., 1999. 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. Kowluru, R.A., Chan, P.S., 2007. Oxidative stress and diabetic retinopathy. Exp. Diabetes Res 2007, 1–14.
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Elevated serum levels of AGEs, sRAGE, and pentosidine in Tunisian patients with severity of diabetic retinopathy Mohsen Kerkeni a,⁎, Amel Saïdi a, Hassan Bouzidi b, Slim Ben Yahya c, Mohamed Hammami a a b c
Laboratory of Biochemistry, UR: Human Nutrition & Metabolic Disorders, Faculty of Medicine, Monastir University, Tunisia Biochemistry Department, CHU Tahar Sfar, Mahdia, Tunisia Ophthalmology Department, CHU Fattouma Bouguiba, Monastir, Tunisia
a r t i c l e
i n f o
Article history: Accepted 17 July 2012 Available online 24 July 2012
a b s t r a c t Objectives: The advanced glycation end products (AGEs)-receptor for AGE (RAGE) axis activity are implicated in diabetic vascular complications. We measured serum AGE, sRAGE and pentosidine levels in Tunisian patients with diabetic retinopathy (DR) and examined whether these biomarkers are related to the severity of DR. Design and methods: We included 30 healthy control subjects and 100 diabetic patients were divided into 2 subgroups: 40 patients with nonproliferative diabetic retinopathy (NPDR), and 60 patients with proliferative diabetic retinopathy (PRD). AGEs, sRAGE and pentosidine were measured in serum by ELISA. Results: Serum levels of AGEs, sRAGE and pentosidine were significantly increased in patients with diabetes mellitus compared to nondiabetic controls (P b .01, P b .001, P b .001 respectively). In diabetic patients, serum AGEs, sRAGE and pentosidine levels were significantly higher in patients who had PDR than in those with NPDR (P = .001, P = .01, P = .005 respectively). Furthermore, in stepwise multivariate regression analysis, the levels of pentosidine and duration of diabetes were independently associated with severity of DR. Conclusion: Serum AGEs, sRAGE, and pentosidine levels are related with the presence of DR. Duration of diabetes and pentosidine were independently correlated with the severity of DR. © 2012 Elsevier Inc. All rights reserved.
Introduction Diabetes mellitus (DM) is increasing at an alarming rate in Tunisian population; patients are prone to the development of macroand microvascular diabetic complications that represent a major cause of morbidity and mortality. DM is now a global pathology, with recent surveys predicting that by 2025, the number of patients with diabetes will rise to staggering 380 million (King et al., 1998; WHO, 2011). This worldwide disease will lead to increasing incidence of the two major types of late complications: macrovasuclar and microvascular that contribute to morbidity and premature deaths. The macrovascular complications, which affect the large vessels include cardiovascular, cerebrovascular, and peripheral vascular disease. The microvascular complications, which affect the small vessels, involve neuropathy, nephropathy, and retinopathy. Diabetic retinopathy (DR) is one of the most significant complications of DM,
Abbreviations: AGEs, Advanced glycation end products; DM, Diabetes mellitus; DR, Diabetic retinopathy; NPDR, Nonproliferative diabetic retinopathy; PRD, Proliferative diabetic retinopathy; sRAGE, soluble form of receptor for advanced glycation end products. ⁎ Corresponding author. E-mail address: [email protected] (M. Kerkeni). 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.mvr.2012.07.006
it occurs in 90% of patients after 20–30 years from the disease diagnosis, and its advanced form, proliferative diabetic retinopathy (PDR), affects over 60% of diabetic patients (Fong et al., 2003). The major risk factors for DR are hyperglycemia, high blood pressure, and duration of diabetes. Chronic hyperglycemia is a major vascular complication of DM. Research worldwide has established various biochemical mechanisms that potentially link hyperglycemia and DR. These pathogenic mechanisms include polyol pathway flux, activation of diacylglycerol (DAG)-PKC pathway, increased expression of growth factors, accelerated formation of advanced glycation end products (AGEs), oxidative stress, hemodynamic or retinal blood flow changes, rennin–angiotensin system activation, and sub-clinical inflammation (Tarr et al., 2011). Recently, AGEs have become more and more focused in this issue. These are formed by the Maillard process, a non-enzymatic reaction between ketones or aldehydes, the amino groups of proteins, lipids and nucleic acids that contributes to the aging of macromolecules (Dyer et al., 1991; Stern et al., 2002; Takeuchi and Yamagishi, 2009). Under hyperglycemic and/or oxidative stress conditions, this process begins with the conversion of reversible Schiff base adducts to more stable, covalently-bound Amadori rearrangement products (Dyer et al., 1991). Over the course of days to weeks, these Amadori products undergo further rearrangement reactions to form the irreversibly-bound moieties known as AGEs. Among
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the few advanced glycation products characterized to date, pentosidine is chemically well defined (Odetti et al., 1992). It is synthesized through nonenzymatic reactions of pentose, and its formation is closely related to oxidative processes (Miyata et al., 1998). Plasma pentosidine was reported to be significantly higher in patients with diabetes than in subjects without diabetes and associated with in an increased incidence of cardiovascular disease and increased stiffening and thickening of the large arteries in patients with type 2 diabetes (Yoshida et al., 2005). Previous studies have confirmed that AGEs and receptor for AGEs (RAGE) interaction elicits oxidative stress generation in various types of cells and subsequently evokes vascular inflammation, macrophage and platelet activation, and thrombosis, thereby playing an important role in the development and progression of vascular complications in diabetes (Bierhaus et al., 1998; Takenaka et al., 2006; Yamagishi et al., 2008a,b). Several different receptors for AGEs have been identified, one of which is termed RAGE. Different cell types including human endothelial cells express RAGE (Yonekura et al., 2003). In addition to cell-bound RAGE, soluble forms of RAGE appear in the plasma, as different splice variants of RAGE (e.g. endogenous secretory receptor for AGE, esRAGE) leaking through the transmembrane and cytosolic domain, and as a proteolytically cleaved form of RAGE (sRAGE), which is most probably shed into the circulation by the sheddase, known as a disintegrin and metalloprotease 10 (ADAM 10) (Hudson et al., 2008; Raucci et al., 2008; Schlueter et al., 2003). The functional role of these soluble forms of RAGE in the circulation remains unclear, but they may reflect the activity of the AGE-RAGE axis. The ligation of RAGE activates the endothelial cell and triggers multiple signaling cascades resulting in activation and translocation of nuclear transcription factors and transcription of the target genes, including adhesion molecules and proinflammatory cytokines (Basta et al., 2002; Harja et al., 2008; Hofmann et al., 1999; Kislinger et al., 1999). Biological function of sRAGE has not been clearly defined and its biological importance within the AGE-RAGE axis is only recently beginning to be understood. In view of these considerations, we have investigated whether AGEs, sRAGE and pentosidine are associated with the presence and the severity of DR; to date little reports have examined the relationship between these three biomarkers and the presence and severity of DR. In the present study, we measured serum AGE, sRAGE and pentosidine levels in Tunisian patients with DR and examined whether these biomarkers are related to the severity of DR. Subjects and methods Subjects The local ethics committee approved this study. Written informed consent was obtained from all patients before the enrollment. In this prospective cohort study, we measured serum concentration of AGEs, sRAGE and pentosidine in 130 participants (age range, 50–75 years). All patients were diagnosed by ophthalmoscopy and fundus stereophotography after dilatation by specialized ophthalmologist in University Hospital Fattouma Bourguiba at Monastir (Tunisia). The severity of the retinopathy was categorized as nonproliferative DR (NPDR, mildto-severe NPDR), or proliferative DR (PDR, level >60). The subjects were divided into 2 groups: Group I is composed of healthy volunteers (n = 30) with no DM or systemic or local eye lesion. Group II is composed of DM patients (n = 100) who developed DR. This group showed DM patient who developed NPDR (n = 40) and DM patient who developed PDR (n = 60). For each patient, a data sheet was completed with the patient's identification code, age, sex, and duration of diabetes, and the antidiabetic agent used, if any. For cardiovascular risk factors, the following definitions were used: individuals were defined as hypertensive if their blood pressure was > 140/90 mm Hg or if they were receiving any antihypertensive treatment; individuals were deemed dyslipidemic when their total cholesterol concentration was
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≥ 5.68 mmol/l, or their triglyceride concentration was ≥2.28 mmol/l, or they receiving lipid-lowering drugs. Methods In all subjects, venous blood was collected in the morning after an overnight fast. The samples were stored at − 80 °C until analysis. Random plasma glucose, hemoglobin A1C (HbA1C) was measured using G7 HPLC Analyser (Tososh Europe N.V), serum creatinine, uric acid, and lipid levels (HDL, LDL, cholesterol, and triglyceride) were measured using enzymatic methods by CX9 Auto-chemical analysis instrument (Beckman CX9, USA). AGEs, sRAGE, and pentosidine were quantitatively determined in serum by enzyme-linked immunosorbent assay (ELISA) kits provided by ABO Switzerland Co., Ltd, according to the manufacturer's instructions. Briefly, the microtiter plate has been pre-coated with an antibody specific to Human AGEs (or sRAGE or Pentosidine). Samples were added, after incubation and washing, plates were incubated with HRP, developed with TMB substrate, and OD450 was determined using an ELISA plate reader. Statistical analysis All values are expressed as mean ± SD. A P value less than 0.05 was considered statistically significant. Significance between 2 groups was determined by independent sample Student t-test for continuous variables. Continuous data from > 2 groups were compared with 1-way analysis of variance (ANOVA). All analyses were performed using the SPSS program (version 17). Correlation was determined by linear regression analysis. Multiple regression was used to further explore the linear relationships between the variables. The equation was in the form: y =a + b1 × x1+ b2 × x2 + … + bp × xp. Regression variables were estimated as well as the correlation coefficient r. ANOVA was used to assess the significance of the regression with significance accepted at P b 0.05. As an alternative data evaluation method, stepwise backward regression analysis was also used on the variables. Results General clinical and biochemical parameters of the studied groups The clinical characteristics and laboratory data of controls and diabetic patients are shown in Table 1. Significant differences were seen between the groups in body mass index, HbA1C, serum glucose, lipids profiles and uric acid but not in serum creatinine. Serum levels of AGEs, sRAGE and pentosidine were significantly increased in diabetic patients than in controls (667.98± 198.51 vs. 508.83 ± 119.68 pg/mL; 206.45 ± 53.18 vs. 148.72 ±32.73 pg/mL; 337.79 ± 63.16 vs. 246.03 ± 55.05 pg/mL, P b 0.01; P b 0.001; and P b 0.001 respectively). Relationship between AGEs, sRAGE, pentosidine levels and severity of DR Table 2 show results of serum AGEs, sRAGE, pentosidine levels in patients with NPDR and patients with PDR and illustrated in Figs. 1, 2 and 3. Patients with PDR showed an increased level of serum AGEs, sRAGE, and pentosidine than patients with NPDR (751.48 ± 209.52 vs. 533.54 ± 82.92 pg/mL; 223.21 ± 62.57 vs. 178.54 ± 11.16 pg/mL; 361.70 ± 66.60 vs. 300.33 ± 36.49 pg/mL, P = 0.001, P = 0.01 and P = 0.005 respectively). Duration of diabetes was higher in patients with PDR than patients with NPDR (P = 0.001). Linear regression and multivariate analyses By linear regression analysis in the 100 diabetic patients with retinopathy, both AGEs and pentosidine levels correlated positively with age, duration of diabetes and serum creatinine (Table 3). sRAGE levels correlated positively with AGEs and creatinine. Furthermore, serum
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Table 1 Clinical characteristics of control subjects and diabetic patients.
N (M/F) Age (years) BMI (kg/m2) Duration of diabetes (years) Hypertension, n (%) Dyslipidemia, n (%) Glucose (mmol/L) HbA1C (%) Total cholesterol (mmol/L) Triglyceride (mmol/L) HDL (mmol/L) LDL (mmol/L) Creatinine (μmol/L) Uric acid (μmol/L) AGEs (pg/mL) sRAGE (pg/mL) Pentosidine (pg/mL)
Control subjects
Diabetic patients
P
30 (17/13) 52 ± 9 25.6 ± 1.2 – 0 (0) 0 (0) 4.77 ± 0.91 5.6 ± 0.2 4.55 ± 0.74 1.42 ± 0.68 1.52 ± 0.47 2.25 ± 0.45 88. 91 ± 15.53 209.06 ± 47.54 508.83 ± 119.68 148.72 ± 32.73 246.03 ± 55.05
100 (53/47) 59 ± 11 29.4 ± 2.4 14.93 ± 8.03 74 (74) 36 (36) 12.35 ± 3.94 8.14 ± 1.6 5.22 ± 0.12 2.15 ± 0.98 1.20 ± 0.31 2.77 ± 0.33 102.42 ± 20.33 287.91 ± 52.80 667.98 ± 198.51 206.45 ± 53.18 337.79 ± 63.16
– NS b 0.05 – b 0.001 b 0.001 b 0.001 b 0.001 b 0.01 b 0.05 b 0.05 b 0.05 NS b 0.01 b 0.01 b 0.001 b 0.001
Values are mean ± SD. BMI: body mass index; NS: not significant.
pentosidine correlated positively with total cholesterol and uric acid. Multiple linear regression was used in patients with PDR to identify any related predictors of severity of DR, evaluating the regression coefficients that represent the contributions of each independent variable to the predictive value of the dependent variable (PDR) (Table 4). This model, which was applied to duration of diabetes, AGEs, sRAGE and pentosidine variables (considered as independent variables), as well as PDR as the dependent variable, showed that duration of diabetes and pentosidine correlated positively.
Discussion Predictors of diabetic complications may be important in the prevention and the management of these complications (van Leiden et al., 2003). The present data demonstrated that duration of diabetes, AGE, sRAGE, and pentosidine levels are positively associated with the presence of DR and duration of diabetes and pentosidine were independently related to PDR. DR is a duration-dependent disease that develops in stages, the incidence of retinopathy is rarely detected in the first few years of diabetes, but the incidence increases to 50% by 10 years and to 90% by 25 years of diabetes (Kowluru and Chan, 2007). As duration of diabetes is a total reflection of blood glucose control and exposing to other risk factors, and the prevalence of DR is highly related with the increasing of HbA1c, duration of diabetes is an important factor for the incidence and development of DR. Similar conclusions were also found in several studies which demonstrated that duration of diabetes and hyperglycemia contributed to the development of DR, and if excellent glycemic control was done at the beginning, DR can be controlled (Herman et al., 1998; McKay et al., 2000). During diabetes, the rate of formation of AGEs exceeds that by first-order kinetics. Thus, over time, even modest hyperglycemic
Fig. 1. Box plots of serum AGEs levels in patients with NPDR and PDR. The horizontal lines in each box plot represent (bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles.
excursions can result in significant adduct accumulation on long-lived macromolecules (Monnier et al., 2005). A number of clinical studies have reported that the formation of AGEs and accumulation have been found in retinal blood vessels of diabetic patients and animals and in human serum and vitreous of diabetic patients which were found to correlate with the degree of DR (Goh and Cooper, 2008). Retinal pericytes which play an important role in the maintenance of microvascular homeostasis have been shown to accumulate AGEs during diabetes which are implicated in endothelial cell injury and blood-retinal barrier dysfunction (Stitt et al., 2000). In addition, AGEs increases vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) expression in microvascular endothelial cells through incellular ROS generation (Yamagishi et al., 2007a). AGEs also activate nuclear factor-B (NF-κB) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase with increase in ROS and apoptosis of pericytes and other retinal cells (Ibrahim et al., 2011; Yamagishi and Matsui, 2011). AGEs disturb microvascular homeostasis through interaction with RAGE. This AGE-RAGE axis plays a central role in the inflammation, neurodegeneration, and microvascular dysfunction in DR (Yamagishi, 2009). A previous study reported that among AGEs in patients with type 2 diabetes, high serum pentosidine is associated with both increased
Table 2 AGEs, sRAGE, and pentosidine levels in diabetic patients with and without PDR patients with DR. Patients with DR
Duration of diabetes (years) HbA1C (%) AGEs (pg/mL) sRAGE (pg/mL) Pentosidine (pg/mL) Values are mean ± SD.
NPDR (n = 40)
PDR (n = 60)
P
8.70 ± 3.16 7.84 ± 1.2 533.54 ± 82.92 178.54 ± 11.16 300.33 ± 36.49
18.29 ± 8.10 8.86 ± 1.5 751.48 ± 209.56 223.21 ± 62.57 361.70 ± 66.60
0.001 0.015 0.001 0.010 0.005
Fig. 2. Box plots of serum sRAGE levels in patients with NPDR and PDR. The horizontal lines in each box plot represent (bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles. Values above the 90th are plotted as points.
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Table 4 Variables estimates for multiple linear regression applied to AGEs, sRAGE, pentosidine and duration of diabetes in diabetic patients with PDR. Term
Estimate
Standard error
t Ratio
P
Intercept AGEs sRAGE Pentosidine Duration of diabetes
56.867 −0.256 −0.048 4.340 21.340
28.336 0.162 0.019 1.182 5.282
2.87 −1.34 −1.37 3.31 3.58
0.034 NS NS 0.0014 0.0010
Co-linearity statistics Tolerance
VIF
0.224 0.243 0.936 0.631
4.628 4.154 1.598 1.098
PDR was the dependent variable; ANOVA revealed a statistically significant fit (P b 0.001) r = 0.755, r2 = 0.570, r2 adjusted = 0.457. VIF: variance inflation factor.
Fig. 3. Box plots of serum Pentosidine levels in patients with NPDR and PDR. The horizontal lines in each box plot represent (bottom to top) the 10th, 25th, 50th (median), 75th, and 90th percentiles. Values below the 10th percentiles are plotted as points.
carotid intima-media wall thickness and arterial stiffening (Yoshida et al., 2005). Recently, Sato et al. reported that plasma pentosidine levels increased significantly in patients with NPDR compared to patients with nondiabetic retinopathy. Authors found that retinal hemodynamic was correlated positively with plasma pentosidine (Sato et al., 2012). Furthermore, Sun et al. recently reported that specific AGE combinations (carboxyethyl-lysine and pentosidine) were strongly associated with complications indicating a link between AGE formation or processing with development of DR (Sun et al., 2011). For pentosidine, as a type of AGE, the study of Salman et al. was done to evaluate the role of pentosidine in predicting the progression of DR. The authors showed that significant elevation of pentosidine was found in patients during the earliest detectable phase of DR (early nonproliferative diabetic retinopathy) and more elevation at the preproliferative stage of retinopathy, returning to lower levels at the proliferative stage of diabetic retinopathy (Salman et al., 2009). However, our results showed that pentosidine levels were increased in diabetic patients with proliferative stage of DR. Indeed, the novelty of this study showed that pentosidine is also a biomarker for severity of DR. Ours current finding seems to agree with the results of a previous study but with small sample size (Ghanem et al., 2011). The present study demonstrated that by multivariate analysis, duration of diabetes and pentosidine was an independent factor increasing the grade of DR. This may reflect declining glomerular filtration rates in association both with aging and duration of diabetes because renal insufficiency may be a major determinant of serum pentosidine. We also found a positive correlation between serum pentosidine and serum creatinine or serum uric acid in diabetic
Table 3 Linear regression analysis of relationships between AGEs, sRAGE, pentosidine levels and characteristics of diabetic patients with retinopathy. Variables
Age (years) BMI Duration of diabetes HbA1C (%) AGEs (pg/mL) Total cholesterol (mmol/L) Triglyceride (mmol/L) Creatinine (μmol/L) Uric acid (μmol/L)
AGEs
sRAGE
Pentosidine
r
P
r
P
r
P
0.321 0.171 0.276 0.133 – 0.122 0.141 0.359 0.121
0.029 NS 0.031 NS – NS NS 0.022 NS
0.152 0.142 0.162 0.052 0.453 0.142 0.126 0.253 0.112
NS NS NS NS 0.008 NS NS 0.039 NS
0.443 0.143 0.353 0.125 0.321 0.243 0.153 0.273 0.253
0.014 NS 0.026 NS 0.029 0.041 NS 0.033 0.039
patients. Because the kidney is the main elimination site for pentosidine (Aso et al., 2004), several studies have examined serum pentosidine concentrations in patients with diabetes with overt nephropathy or those with chronic renal disease, reporting elevations (Miyata et al., 1996; Sugiyama et al., 1998; Suliman et al., 2003). Especially, a dramatic increase in plasma pentosidine was reportedly found in patients with end-stage renal disease (Weiss et al., 2000). Renal insufficiency thus may lead to accumulation of pentosidine in the blood because renal is the major determinant of serum pentosidine (Hricik et al., 1993). Formation of pentosidine requires oxidation as well as glycation. It might promote oxidative stress and endothelial cell dysfunction and induce the growth and tube formation of microvascular endothelial cells through autocrine vascular endothelial growth factor. We speculated that AGEs and pentosidine might accelerate the development and severity of DR both by accumulation of AGEs in the vessel walls and by causing endothelial dysfunction, as mediated by the AGE-RAGE axis activity. Moreover, we speculated that increased pentosidine might be involved in the stiffening of the retinal arteries. The increased levels of sRAGE across stage of severity of retinopathy observed in our study were also in line with following observations: (a) circulating sRAGE levels are increased, rather than decreased, in both type 1 and type 2 diabetic patients (Challier et al., 2005; Tan et al., 2006); (b) serum sRAGE levels are positively, but not inversely, correlated with circulating AGE levels in both non-diabetic and type 2 diabetic subjects (Tan et al., 2006; Yamagishi et al., 2006); (c) AGEs are positive regulators of cell surface expression of RAGE (Tanaka et al., 2000; Yamagishi and Takeuchi, 2004; Yamagishi et al., 2007b) and that RAGE is up-regulated in atherosclerostic plaques in diabetes, diabetic nephropathy and retinopathy (Cipollone et al., 2003; Pachydaki et al., 2006; Tanji et al., 2000); (d) vitreous levels of sRAGE are increased in proliferative retinal diseases by reflecting enhanced RAGE expression in epiretinal membranes of the eyes (Pachydaki et al., 2006). Treatment for reducing AGEs also prevents the onset or progression of DR. Because the current study had a small sample size, another clinical study of correlation between these biomarkers and the severity of DR is needed. In summary, the current study showed that serum AGEs, sRAGE and pentosidine levels increased in Tunisian patients with DR and duration of diabetes and pentosidine was independently correlated with severity of DR. Our results suggest that increased AGE-RAGE activity and increased of pentosidine may initiate and extent the severity of diabetic retinopathy. Intervention on serum AGEs, sRAGE and pentosidine may be a potential therapy for diabetes and its complications.
Conflict of interest statement The authors declare that they have no conflict of interest.
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