Glyoxalase-1 overexpression reduces endothelial dysfunction and attenuates early renal impairment in a rat model of diabetes
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In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed the MGO-detoxifying enzyme glyoxalase-I (GLO-I), to determine the impact of intracellular glycation on vascular function and the development of early renal changes in diabetes.
Wild-type and Glo1-overexpressing rats were rendered diabetic for a period of 24 weeks by intravenous injection of streptozotocin. Mesenteric arteries were isolated to study ex vivo vascular reactivity with a wire myograph and kidneys were processed for histological examination. Glycation was determined by mass spectrometry and immunohistochemistry. Markers for inflammation, endothelium dysfunction and renal dysfunction were measured with ELISA-based techniques.
Diabetes-induced formation of AGEs in mesenteric arteries and endothelial dysfunction were reduced by Glo1 overexpression. Despite the absence of advanced nephrotic lesions, early markers of renal dysfunction (i.e. increased glomerular volume, decreased podocyte number and diabetes-induced elevation of urinary markers albumin, osteopontin, kidney-inflammation-molecule-1 and nephrin) were attenuated by Glo1 overexpression. In line with this, downregulation of Glo1 in cultured endothelial cells resulted in increased expression of inflammation and endothelium dysfunction markers. In fully differentiated cultured podocytes incubation with MGO resulted in apoptosis.
This study shows that effective regulation of the GLO-I enzyme is important in the prevention of vascular intracellular glycation, endothelial dysfunction and early renal impairment in experimental diabetes. Modulating the GLO-I pathway therefore may provide a novel approach to prevent vascular complications in diabetes.
KeywordsEndothelial dysfunction Glycation Glyoxalase-1 Methylglyoxal Renal impairment
Advanced glycation end-products
Endothelial nitric oxide synthase
Intercellular adhesion molecule-1
Kidney injury marker-1
Mean arterial pressure
Monocyte chemotactic protein-1
Nuclear factor (erythroid-derived 2)-like 2
Ultra-performance liquid chromatography tandem mass spectrometry
Vascular cell-adhesion molecule-1
Hyperglycaemia is a key risk factor for microvascular complications leading to excess morbidity and mortality in diabetic patients . Microangiopathy, damage to the small blood vessels and capillaries, is a direct result of chronic hyperglycaemia and is the main cause of major complications such as retinopathy and nephropathy [2, 3]. Dysfunction of the vascular endothelium is considered to be an important factor in the initiation, progression and clinical outcome of diabetic microangiopathy . One hypothesis as to how hyperglycaemia leads to endothelial dysfunction and, consequently, vascular complications is the formation of advanced glycation end-products (AGEs) .
AGEs are a heterogeneous family of non-enzymatically modified proteins, which are increased in patients with diabetes. In addition to the formation of AGEs by carbohydrates in the classical Maillard reaction, intracellular glucose-derived glycolytic intermediates, such as methylglyoxal (MGO), glyoxal (GO) and 3-deoxyglucosone (3-DG), also form AGEs . Because these intracellular dicarbonyl compounds form many more glycated proteins and at a faster rate than do equimolar amounts of glucose, intracellular AGE formation is thought to play an important role in the link between AGEs and diabetic complications. Indeed, intracellular AGEs have been implicated in activating intracellular signalling pathways as well as in modifying the function of intracellular proteins, thereby contributing to diabetic vascular complications . The very reactive oxo-aldehyde MGO has been identified as the major precursor in the formation of intracellular AGEs in endothelial cells . MGO is formed mainly by fragmentation of the triose phosphates glyceraldehyde-3-phosphate and dihydroxy-acetone-phosphate and is, under physiological circumstances, efficiently detoxified to d-lactate by the glyoxalase system, in which glyoxalase-I (GLO-I) is the rate limiting enzyme . MGO is known to have detrimental effects on cellular function and it is also evident that elevated levels of MGO are responsible for renal oxidative stress, as demonstrated in diabetic rats . Most importantly, progression of hypertension and diabetic nephropathy in humans is also significantly related to increased levels of MGO [11, 12].
We previously showed in a rat model of diabetes that Glo1 overexpression detoxifies MGO and GO, and thereby decreases AGEs and markers of oxidative stress . Furthermore, we recently demonstrated in an ex vivo model that high glucose levels and short-term diabetes-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries are mediated by intracellular MGO levels . In addition, Glo1 overexpression protects against diabetes-induced retinal neuroglial and vasodegenerative pathology . However, the involvement of the major AGE precursor MGO in long-term diabetes-induced endothelial dysfunction and, consequently, the development of renal microvascular complications remain largely unknown.
Therefore, the present study explored the effect of Glo1 overexpression on microvascular and renal function 24 weeks after streptozotocin (STZ)-induced diabetes in the rat. We hypothesised that elevated levels of MGO are involved in microvascular dysfunction and in the development of diabetic renal changes, and that Glo1 overexpression can prevent this.
Heterozygous transgenic Glo1-overexpressing rats were crossed with wild-type Wistar rats (Nippon Seibutsu Zairyo Center, Saitama, Japan) to obtain enough Glo1-overexpressing progeny for the experiment . Young adult wild-type and Glo1 transgenic male rats (10 weeks of age) were rendered diabetic for a period of 24 weeks by a single intravenous injection of STZ (Sigma-Aldrich, Zwijndrecht, the Netherlands; 45 mg/kg body weight). Only rats with fasting blood glucose levels above 15 mmol/l were included in the study (Contour test strips, Bayer Health Care, Leverkusen, Germany). Weight- and age-matched control wild-type rats were not injected. The rats were divided into three groups: wild-type control group (WtC; n = 9), wild-type STZ-induced diabetic group (WtD; n = 8) and Glo1-overexpressing STZ-induced diabetic group (TgD; n = 13).
The study protocols were approved by the Committee on Animal Experimentation and Welfare of Maastricht University.
Twenty-four weeks after the induction of diabetes, rats were killed and mesenteric arteries, kidneys, urine and EDTA blood were processed for further analysis.
Blood pressure and heart rate measurement
Under isoflurane (Baxter, Deerfield, IL, USA) anaesthesia a PE-10 catheter was inserted in the abdominal aorta via the femoral artery and connected to a pressure transducer (Miller Instruments, Houston, TX, USA). The pressure signal was digitally sampled at 2 kHz and mean arterial pressure (MAP) and heart rate were calculated over a 10–15 min time period after haemodynamic variables were stabilised.
Measurement of GLO-I activity
GLO-I activity was assayed by spectrophotometry (Synergy, BioTek, Winooski, VT, USA), by monitoring the increase in absorbance at 240 nm due to the formation of S-d-lactoylglutathione for 10 min at 37°C according to the method of McLellan and Thornalley .
Measurement of vascular function, morphology and gene expression
On paraformaldehyde-fixed mesenteric arteries, media cross-sectional area, media thickness and lumen diameter were determined using Leica Qwin software (Leica, Groot Bijgaarden, Belgium) on 4 μm sections stained with Lawson’s solution (Boom, Meppel, the Netherlands), a classic elastin stain. To stain collagen, cross-sections were deparaffinised and incubated in phosphomolybdenic acid (0.2% wt/vol.) for 5 min, followed by incubation with Sirius red (Sigma-Aldrich). After washing with 0.1 mol/l HCl for 2 min, sections were dehydrated and protected with coverslips. Collagen content was calculated as the percentage collagen-positive area per total area.
After homogenisation of vascular tissue by the use of glass beads and a mini-bead beater, total cellular RNA was extracted using TRIzol isolation (Invitrogen, Life Technologies, Paisley, UK). Reverse transcription was performed with a Reverse Transcriptase kit from Invitrogen. Subsequently, quantitative PCR (qPCR) assays were performed using Quanta Universal PCR MasterMix on an ABI PRISM 7900 HT (Applied Biosystems, Foster City, CA, USA) and a primer of the target (i.e. vascular cell-adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], endothelial nitric oxide synthase [eNOS]) or a primer of the housekeeping gene, β-actin, all purchased from Eurogentec (Liège, Belgium).
Immunohistochemical and histological examinations
Mesenteric arteries and kidneys were fixed with phosphate-buffered (pH 7.4) formaldehyde (4% vol./vol.) overnight at room temperature. Subsequently, arteries and kidneys were transferred to 70% vol./vol. ethanol, embedded in paraffin and processed for histological examination.
Anti-GLO-I (1:1,000; see ESM Methods), anti-N ε-(1-carboxymethyl)lysine (CML) (1:4,000; homemade) , anti-N δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-l-ornithine (MG-H1) (1:100,000, generous gift from M. Brownlee), anti-eNOS (1:5,000; BD transduction laboratories, Breda, the Netherlands), anti-Wilms’ tumour-1 (WT-1) (undiluted; DAKO, Glostrup, Denmark), and anti-active-caspase-3 (1:200; Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. After washing in PBS (pH 7.4), sections were incubated for 30 min with the appropriate biotin-labelled antibody (1:500) at room temperature and subsequently washed in PBS. After incubation with streptavidin–horseradish peroxidase (1:200, DAKO) for 60 min at room temperature, peroxidase was visualised with 3,3-diamino-benzidine-tetrahydrochloride/H2O2 (Sigma-Aldrich) for 3–5 min. Immunostaining was scored for anatomical localisation and its intensity or number of positive cells. For the intensity scoring, each positive vessel was given a score: 0, absent; 1, weak positivity; 2, moderate positivity; 3, strong positivity. Each multiplication score was then added and the sum was divided by the number of samples, resulting in an immunohistochemical score. All stainings were scored by two blinded observers.
The middle part of the right kidney (containing the papilla) was embedded in paraffin for examination. Two micron-thick sections were cut on a rotation microtome and stained with p-aminosalicylic acid and haematoxylin (DAKO). Total glomerulus volume was measured observer-blinded at a magnification of ×400 as previously described .
Creatinine measurement in plasma and urine
Urinary and plasma creatinine levels were measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS) (UPLC Acquity and Micromass Quattro Premier XE Tandem Mass Spectrometer; Waters, Milford, MA, USA) as described elsewhere . Internal standard creatinine (methyl-d3) (CDN Isotopes, Quebec, Canada) was mixed with deproteinised plasma or diluted urine and subsequently analysed by UPLC-MSMS.
Rat kidney injury panel 1 was purchased from Meso Scale discovery (Rockville, MD, USA). This assay detects lipocalin-2, osteopontin and kidney-injury-molecule (KIM-1) in a sandwich immunoassay and uses a competitive assay format to detect albumin. Each 96-well plate had four carbon electrodes in the bottom of each well; each pre-coated with one of the four anti-kidney-injury marker antibodies of interest. The intra-assay variations of albumin, lipocalin-2, osteopontin and KIM-1 were 5.0%, 4.0%, 8.4% and 3.3%, respectively. Monocyte chemotactic protein-1 (MCP-1) was measured with MSD kit K151AYB-2 (Meso Scale) according to the manufacturer’s protocol (intra-assay variation was 3.3%). The markers ICAM-1 (Diaclone, Ann Arbor, MI, USA), IL-6 (Sanquin, Amsterdam, the Netherlands) and nephrin (Exocell, Philadelphia, PA, USA) were measured using standard ELISA procedures (according to the manufacturer’s protocol) and the intra-assay variations were 2.8%, 1.6% and 9.1% respectively.
Plasma nitrite and nitrate levels were determined using a commercially available colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) based on the Griess reaction. Before the assay, the plasma samples were ultrafiltered though a 30 kDa molecular mass cut-off filter (Ultrafree-MC centrifugal filter units; Millipore Corporation, Bedford, MA, USA) to reduce the background absorbance caused by haemoglobin.
Measurement of AGEs and oxo-aldehydes
The AGEs CML, N ε-(1-carboxyethyl)lysine (CEL) and MG-H1 were measured using UPLC-MSMS (Waters) as described earlier . Oxo-aldehydes GO, MGO and 3-DG in plasma were assayed by derivatisation with o-phenylenediamine and UPLC-MSMS .
In vitro cell culture experiments
Human immortalised endothelial cells (ECRF-24) were cultured for Glo1 silencing experiments and conditionally immortalised mouse podocytes were cultured for apoptosis assays. For further details see ESM Methods.
All values are expressed as mean ± SEM and p < 0.05 was considered statistically significant. For further details see ESM Methods.
General physical and biochemical characteristics
General physical and biochemical characteristics of the rats after 24 weeks of diabetes
Plasma glucose (mmol/l)
6 ± 1
32 ± 1*
31 ± 1*
Body weight (g)
475 ± 12
300 ± 11*
300 ± 12*
Food intake (g)
19 ± 2
43 ± 2*
43 ± 2*
Fluid intake (ml/day)
19 ± 2
187 ± 14*
200 ± 13*
Urine production (ml/day)
11 ± 1
183 ± 15*
207 ± 11*
0.40 ± 0.03
0.81 ± 0.10*
0.66 ± 0.02†
0.33 ± 0.02
0.64 ± 0.07*
0.50 ± 0.02†
3.2 ± 0.1
6.1 ± 0.2*
6.1 ± 0.2*
Urinary d-lactate (μmol/mmol creatinine)
1 ± 0.3
196 ± 63*
568 ± 237***
4.8 ± 0.5
10.8 ± 1.1*
10.4 ± 1.2*
7.5 ± 0.8
15.0 ± 1.4*
13.5 ± 1.4*,†
206 ± 10
244 ± 11*
199 ± 9†
91 ± 2
62 ± 2*
76 ± 3*,†
Heart rate (bpm)
340 ± 12
261 ± 4*
284 ± 9*
Both MAP and heart rate were decreased in the wild-type diabetic rats compared with the control rats (Table 1). The decrease in blood pressure after 24 weeks of diabetes was significantly attenuated by Glo1 overexpression, although heart rate was not statistically influenced by the overexpression of Glo1.
Diabetes-induced AGE formation in rat mesenteric arteries is prevented by Glo1 overexpression
Morphological analyses of the mesenteric arteries showed no differences in mesenteric lumen radius, media cross-sectional area or media thickness after 24 weeks of diabetes (see ESM Table 1). Analyses of the mesenteric arteries for collagen deposition also showed no significant differences in collagen content corrected for medial area between the different groups.
Diabetes-induced impairment of vasorelaxation in rat mesenteric arteries is reduced by Glo1 overexpression
Modulation of GLO-I activity regulates the expression of endothelial activation and inflammation markers
Markers of early renal damage are attenuated by Glo1 overexpression after 24 weeks of diabetes
Renal characteristics of the rats after 24 weeks of diabetes
Left kidney weight (g)
1.4 ± 0.1
2.0 ± 0.1*
1.8 ± 0.1*
Right kidney weight (g)
1.5 ± 0.1
2.0 ± 0.1*
1.8 ± 0.1*
Left kidney/body weight ratio (mg/g)
3.0 ± 0.1
6.6 ± 0.2*
6.1 ± 0.1*
Right kidney/body weight ratio (mg/g)
3.1 ± 0.1
6.5 ± 0.2*
6.1 ± 0.2*
Glomerular volume (mm3)
86 ± 8
129 ± 14*
103 ± 16
Creatinine clearance (ml h−1 [100 g]−1)
56 ± 6
103 ± 40
71 ± 6
Immunohistochemical analyses showed increased presence of CML and MG-H1 in the mesangial matrix and peritubular capillaries of the diabetic rats, which could not be prevented by Glo1 overexpression. This observation was also confirmed by UPLC-MSMS analysis of total kidney lysates (data not shown). Furthermore, extensive histological analysis of the kidney did not show any collagen deposition or fibrotic or inflammatory differences in glomerular, tubulointerstitial or capillary tissue between the three groups after 24 weeks of diabetes (ESM Fig. 1).
In line with the in vivo data, we observed that in fully differentiated cultured podocytes incubation with 1,000 μmol/l MGO for 48 h resulted in an increase in apoptotic cells compared with control cells (Fig. 5j). Early and late apoptotic cells were quantified with annexin-V and propidium iodide FACS, respectively (Fig. 5k). These data indicate that the apoptosis-induced decrease in podocytes in the diabetic glomerulus can at least be partially caused by MGO cytotoxicity.
In this study we showed that diabetes-induced impairment of vascular function, as measured by an impaired endothelium-dependent relaxation and an increased expression of markers for endothelial dysfunction, in mesenteric arteries of diabetic rats is prevented by overexpression of Glo1. In line with this, downregulation of Glo1 in cultured human endothelial cells resulted in a pro-inflammatory and endothelial-activated profile. In addition, overexpression of Glo1 attenuated loss of podocytes in the glomerulus and also renal excretion of early markers of diabetic nephropathy. MGO-induced podocyte apoptosis and generation of AGEs in the vasculature seem to contribute to this pathogenesis. These data demonstrate a direct link between increased levels of AGEs, endothelial dysfunction and the development of renal changes in experimental diabetes.
Our results showed that the diabetes-induced elevation of the AGEs CML and MG-H1 in vascular tissue is prevented by overexpression of Glo1. This decrease in vascular tissue levels of MGO and GO-derived AGEs demonstrates the efficiency of Glo1 overexpression in the detoxification of both GO and MGO. This is in accordance with our previous study in which we demonstrated that Glo1 overexpression prevents elevated blood levels of GO and MGO, and thereby decreases plasma AGE levels after 12 weeks of diabetes . In contrast, the CML levels in the plasma could not be significantly decreased by Glo1 overexpression after 24 weeks of diabetes, suggesting that an excessive formation of CML via additional pathways, like lipid peroxidation, possibly exceeds the detoxification rate in this prolonged state of diabetes.
We previously addressed the reaction of the mesenteric endothelium to acetylcholine after short-time incubations with MGO or high glucose and in diabetic rats 12 weeks after STZ injection . In the current study we confirmed these data in mesenteric arteries after 24 weeks of diabetes and extended our observations by specifically addressing which pathways are involved in the diabetes-induced impairment of acetylcholine-dependent relaxation. Our approach revealed that the decreased endothelium-dependent relaxation is caused by impairment of both the eNOS/NO pathway and endothelium-derived hyperpolarising factors. In contrast with several studies suggesting a decrease in expression and activity of the eNOS enzyme [22, 23], our results show that its mRNA and protein levels were not altered by diabetes. Unfortunately, we did not address the phosphorylation state of eNOS, therefore we could not attribute the impairment of eNOS activity to decreased phosphorylation, as Dhar et al described . However, we previously showed that MGO and MGO-arginine adducts do not directly inhibit the overall eNOS activity in vitro . Furthermore the plasma levels of the NO metabolites nitrite and nitrate were significantly decreased in the wild-type diabetic rats, indicating a decrease in NO bioavailability rather than reduced NO production. Interestingly, mRNA levels of the genes encoding oxidative stress-sensing master switch nuclear factor (erythroid-derived 2)-like 2 (NRF-2), and NRF-2-responsive detoxifying genes Glo1, NADPH-oxidase-4 and sodium oxide dismutase-3, were increased in the wild-type diabetic rats (data not shown), and this oxidative stress response was absent in the GLO-I diabetic rats, suggesting that diabetes-induced quenching of NO by oxidative stress occurred.
Elevated levels of cellular adhesion molecules are associated with an increase in leucocyte adhesion and inflammatory activation . Our results showed that Glo1 overexpression prevented the STZ-induced increase of both Vcam1 and Icam1 expression. In line with this observation, our experiments in cultured endothelial cells showed that GLO-I is an important regulator in the prevention of endothelial inflammation and activation. These results confirmed the link between markers of endothelial function and AGEs [27, 28].
Diabetes is characterised by hypertrophy of several target tissues including the kidneys and the vasculature [29, 30]. Consistent with these findings, we detected an increase in kidney weight and glomerular size after 24 weeks of diabetes. Overexpression of Glo1 revealed a trend of decreasing the renal hypertrophy, although not statistically significant. We did not see any differences in morphological lesions of the glomeruli even after 24 weeks of diabetes, which is in accordance with earlier studies in STZ rats. Hirose et al  reported that basal membrane thickness is the only variable of advanced diabetic glomerulopathy that increases significantly in STZ rats, albeit only after more than 6 months of diabetes. Bidani et al  speculated that blood pressure reduction in STZ rats is one of the reasons that the nephrotic damage observed in the STZ diabetes model is only modest. Indeed, in our model we also observed a decrease in blood pressure in the diabetic rats and, interestingly, Glo1 overexpression significantly blunted the diabetes-induced blood pressure depression.
In humans, there is convincing evidence that endothelial dysfunction is closely associated with the development of diabetic nephropathy [33, 34]. In type 1 diabetes, microalbuminuria and macroalbuminuria are accompanied by a variety of markers of endothelial dysfunction, such as increased plasma concentrations of VCAM-1, endothelin-1 and plasminogen activator inhibitor-1, and an impaired endothelium-dependent, NO-mediated vasorelaxation . Consistent with this, we observed an increase in albuminuria in diabetes, which showed a tendency to be improved by Glo1 overexpression. In addition to albumin (which was not statistically significant), two other markers for nephropathy, KIM-1 and osteopontin, were significantly improved by Glo1 overexpression. Although the function of KIM-1 is unclear, its abundant tubular expression after damage points to a role in either tubular damage or repair . Also osteopontin, a large phosphoglycoprotein adhesion molecule, is upregulated in diabetic nephropathy. A recent study by Nicholas et al suggests that osteopontin is a key profibrotic factor that contributes to the development of glomerulosclerosis and albuminuria in diabetes .
Diabetic nephrotic injuries of both the glomeruli and the tubulointerstitium are accompanied by increased protein excretion and consequently by a decline in renal function . It is likely that diabetic nephropathy occurs as a result of an interplay between haemodynamic and metabolic factors . Thus, endothelial dysfunction, as an early phenomenon, may explain the typical association between albuminuria and extra-renal complications. It may be speculated that endothelial dysfunction, as we observed in the rat mesenteric arteries, is also present in the intra-renal vasculature [40, 41], thereby resulting in increased pressure in the glomeruli, which consequently leads to glomerular dysfunction and urinary protein leakage. Our observations that Glo1 overexpression reduced diabetes-induced vascular and renal dysfunction, as well as data from other studies [30, 42, 43], support a role for glycation in these pathogeneses. In addition, a cohort study by Beisswenger et al showed that progression of diabetic nephropathy is also significantly related to elevation in dicarbonyl stress . In this study elevated levels of MGO correlated with loss of podocytes, another mechanism preceding glomerular leakage . This strengthens our observations that MGO induces podocyte apoptosis and that diabetes-induced podocyte loss can be prevented by Glo1 overexpression.
In summary, this study showed for the first time that overexpression of Glo1 reduces diabetes-induced endothelial dysfunction and attenuates early renal impairment in an animal model of diabetes. These observations suggest that inhibition of intracellular glycation prevents deterioration of vascular and renal function in experimental diabetes. A recent study with the angiotensin type 1 receptor blocker candesartan showed that GLO-I activity can be positively regulated by drug intervention . Therefore activation of GLO-I may provide an approach to prevent endothelial dysfunction and vascular complications in diabetes.
The authors gratefully acknowledge M. van den Waarenburg and J. Scheijen (Department of Internal Medicine, Division of General Internal Medicine, Laboratory for Metabolism and Vascular Medicine, Maastricht University, Maastricht, the Netherlands) for the measurement of the urinary kidney injury markers, soluble adhesion molecules and plasma/tissue AGE levels. Furthermore we also acknowledge J. Debets (Department of Pharmacology, Maastricht University, Maastricht, the Netherlands) for the measurement of intra-arterial blood pressure in the rats.
This study was partially supported by the Dutch Diabetes Foundation (Grant number 2005.11.013) and further supported by the Swiss National Science Foundation Fellowship PBBSP3-144160 (JS), National Institutes Health Grants DK62472 and DK57683 (PM) and the Danish Diabetes Academy supported by the Novo Nordisk Foundation (JAØ).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
OB, AF, CJPK, BJAJ, JGRDM and CGS designed the study and experiments. TM, MB, PHM and CDAS contributed experimental and conceptual expertise. OB, PMGN, JAØ, and JS performed the research. All authors analysed, interpreted and discussed the data. OB and CGS wrote the manuscript. All authors reviewed and revised the manuscript critically. All authors approved the final version of the manuscript.
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