Nε-(Carboxymethyl)lysine Depositions in Intramyocardial Blood Vessels in Human and Rat Acute Myocardial Infarction
A Predictor or Reflection of Infarction?
Objective— Advanced glycation end products (AGEs), such as Nε-(carboxymethyl)lysine (CML), are implicated in vascular disease. We previously reported increased CML accumulation in small intramyocardial blood vessels in diabetes patients. Diabetes patients have an increased risk for acute myocardial infarction (AMI). Here, we examined a putative relationship between CML and AMI.
Methods and Results— Heart tissue was stained for CML, myeloperoxidase, and E-selectin in AMI patients (n=26), myocarditis patients (n=17), and control patients (n=15). In AMI patients, CML depositions were 3-fold increased compared with controls in the small intramyocardial blood vessels and predominantly colocalized with activated endothelium (E-selectin–positive) both in infarction and noninfarction areas. A trend of increased CML positivity of the intima of epicardial coronary arteries did not reach significance in AMI patients. In the rat heart AMI model, CML depositions were undetectable after 24 hours of reperfusion, but became clearly visible after 5 days of reperfusion. In line with an inflammatory contribution, human myocarditis was also accompanied by accumulation of CML on the endothelium of intramyocardial blood vessels.
Conclusions— CML, present predominantly on activated endothelium in small intramyocardial blood vessels in patients with AMI, might reflect an increased risk for AMI rather than being a result of AMI.
Advanced glycation end products (AGEs) are advanced products of the Maillard reaction, including pentosidine, Nε-(carboxyethyl)lysine, and Nε-(carboxymethyl)lysine (CML). AGEs also accumulate during aging1,2 and at an accelerated rate in diabetes.3,4 In diabetes patients, the accumulation of AGEs has been associated with vascular complications.5,6 AGEs are also found in human atherosclerotic lesions in blood vessels, raising a potential link between deposition of AGEs and atherogenesis.7
The major AGE CML has received considerable interest, because it can act as a ligand for the receptor of AGE (RAGE) and it has been associated with increased oxidative stress.8 CML can be formed on proteins by an oxidative cleavage of the Amadori product fructose-lysine,9,10 and by a reaction of proteins with the peroxidation products of polyunsaturated fatty acids11 or the dicarbonyl compound glyoxal.12,13 Recent data indicated that myeloperoxidase activity and NADPH oxidase can also represent an important source of CML in tissue proteins.14 It has been demonstrated that CML is enhanced in vascular tissue of diabetic patients and in human atherosclerotic lesions.15,16 We recently demonstrated in diabetic patients without AMI or any form of cardiomyopathy an increased deposition of CML in small intramyocardial blood vessels of the heart that were without morphological changes.17
While studying the pathophysiological role of CML depositions in the heart in more detail, we analyzed CML depositions in heart tissue of AMI patients and observed clear CML deposition in the intramyocardial blood vessels of these patients. Subsequently, the role and mechanism of generation of these CML depositions were further analyzed in an in vivo model of AMI in rats, at a cellular level in endothelial cells, and in human heart tissue of patients with myocarditis.
Patients included in this study underwent autopsy within 24 hours after death. We included heart tissue from 15 control patients and 26 patients with AMI (Table). The AMI patients had infarcts of variable duration. From these AMI patients, tissue was taken from the infarcted as well as the noninfarcted areas of the heart. In addition, epicardial coronary artery sections were taken. Patients with diabetes were excluded because theoretically this condition can also cause increased CML depositions. Control patients died because of a cause not related to any form of heart disease and did not have inflammation of the heart. In addition, we included heart tissue from 17 patients with myocarditis (bacterial myocarditis n=4; viral myocarditis n=13) without signs of other forms of heart disease. Age and sex distribution did not differ significantly between the control or AMI group. From all patients included in this study, tissue from lung and kidney also were obtained for immunohistochemical analysis.
This study was approved by and performed according to the guidelines of the ethics committee of the VU University Medical Center, Amsterdam. Use of leftover material after the pathological examination has been completed is part of the patient contract in our hospital.
mAb Against CML
In a recent study we described the development and characterization of a specific CML monoclonal antibody (mAb).18 Briefly, antisera against CML-modified keyhole limpet hemocyanin were raised in mice (Harlan, Horst, the Netherlands), and the mouse with the highest titer for CML-human serum albumin (HSA) was used for the production of mAbs. One of these, of the IgG1 class, was used in this study. The antibody immunoreactivity for CML-HSA, as determined in enzyme-linked immunosorbent assay (ELISA), appeared to be proportional to the yield of CML as determined using stable-isotope dilution tandem mass spectrometry (LC-MSMS), as recently described in detail.19 The antibody did not cross-react with Nε-(carboxyethyl)lysine.
For immunohistochemistry paraffin-embedded tissue sections (4 μm) were used. After deparaffinization and dehydration, sections were stained with hematoxylin-eosin and subsequently incubated with 0.3% H2O2 in methanol for 30 minutes. Sections were not heated to prevent artificial induction of CML by this procedure.9 After incubation with normal rabbit serum (1:50; Dako, Glostrup, Denmark) for 10 minutes, sections were incubated for 60 minutes with anti-CML (1:500) or anti- myeloperoxidase (MPO) (1:500; mAb, Dako). After washing in phosphate-buffered saline (PBS), pH 7.4, sections were incubated for 30 minutes with rabbit anti-mouse biotin-labeled antibody (1:500; Dako), washed in PBS, incubated with streptavidin-horseradish peroxidase (HRP) (1:200; Dako) for 60 minutes, and visualized with 3,3-diamino-benzidine-tetrahydrochloride/H2O2 (DAB) (Sigma Chemical Company, St. Louis, Mo) for 3 to 5 minutes. For E-selectin, sections were incubated for 30 minutes in pepsin and stained with anti-E-selectin (1:50; mAb, Monosan, Uden, The Netherlands). After washing in PBS, sections were incubated for 60 minutes with rabbit-anti-mouse-HRP (1:1000; Dako), subsequently washed in PBS, and then visualized with DAB for En Vision (Dako) (3 to 5 minutes).
Microscopic criteria20–22 were used to estimate infarct duration in all myocardial tissue specimens.
Immunoscoring was performed by 3 independent investigators (A.B., P.A.J.K., and H.W.M.N.). CML, MPO, and E-selectin positivity were scored for anatomic localization and intensity. For the intensity scoring each positive vessel was given a score of: 1=weak positivity; 2=moderate positivity; or 3=strong positivity. CML positivity in coronary arteries was measured as the area of CML positivity/ mm2 intima using Q-prodit.23 Subsequently, the area of the slide was measured. Each intensity score was multiplied by the amount of vessels positive for this score. Each multiplication score (for 1, 2, and 3) was then added and the sum subsequently was divided by the slide area resulting in a immunohistochemical score per cm2.
The HUVEC-derived immortalized EC-RF24 endothelial cells24 were cultured on fibronectin-coated, tissue-culture plates in “growth medium” containing medium-199 (Gibco-BRL, Gaithersburg, Md) supplemented with 20% (v/v) fetal bovine serum, 2 mmol/L L-glutamine, 50 μg/mL heparin (Sigma), 12.5 μg/mL EC growth supplement (Sigma), and 100 U/mL penicillin/streptomycin (Gibco-BRL) under a 5% CO2 atmosphere at 37°C. Metabolic inhibition was induced by incubating the cells in 20 mmol/L 2-deoxy-d-glucose (Sigma, St. Louis, Mo) in PBS for 2 hours under a 5% CO2 atmosphere at 37°C. Oxidative stress was induced by incubation in 0.03% H2O2 in PBS for 2 hours under a 5% CO2 atmosphere at 37°C. Cells were then reperfused for 24 hours with normal culture medium.
EC-RF24 cells were dissolved in sodium dodecyl sulfate sample buffer, stirred, and heated for 10 minutes at 95°C. The samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with CML antibody (1/1000 dilution) and subsequently with horseradish peroxidase conjugated rabbit-anti-mouse immunoglobulins (RaM-HRP; Dakopatts, Glostrup, Denmark; 1/1000 dilution). The blots were then visualized by enhanced chemiluminescence (ECL) (Amersham, Buckinghamshire, UK). As a positive control CML-modified albumin was used.
AMI in the Rat
Female Wistar rats (7 to 8 weeks of age, Harlan CPB, Zeist, The Netherlands) were acclimated to the facility for at least 2 weeks before surgery. Rats were anesthetized with halothane (with O2 and N2O in a 2:1 ratio), endotracheally intubated, and ventilated by a respirator. After a left-sided thoracotomy, the left coronary artery was occluded ≈2 mm from the origin with a 7-0 silk suture. Thirty minutes after ligation, the ligature was released for reperfusion for different periods of time, varying from 2 up to 24 hours, and 5 days.
Infarct areas were determined using Nitro Blue Tetrazolium. Subsequently, sections were made for immunohistochemical detection of CML.
All animals were treated in compliance with the Dutch guidelines for the care and use of laboratory animals and the experiments were approved by the institutional ethical committee for animal experimentation.
Peritoneal Dialysis of the Rat
Rats (n=5) received 10 mL lactate buffered 3.86% glucose-containing peritoneal dialysis fluid during a 5-week period via a subcutaneously implanted mini access port that was connected via a catheter to the peritoneal cavity. Subsequent to euthanizing the rats, peritoneal tissue was isolated and CML on this tissue was determined by immunohistochemistry.
Data were analyzed with SPSS for windows version 9.0. To evaluate whether observed differences were significant, Mann-Whitney analysis, independent t tests or χ2 tests were used when appropriate. In the text and relevant figure, values are given as means±standard error (SE). A 2-sided P<0.05 was considered to represent a significant difference.
CML Accumulates in Intramyocardial Blood Vessels of AMI Patients
The presence of CML in human heart tissue was investigated in tissue specimens of AMI patients and in control patients. Immunohistochemical analysis revealed no or only focal weak staining of CML in especially endothelial cells of intramyocardial blood vessels in control patients (Figure 1A). Notably, these control patients had different degrees of atherosclerotic lesions, varying from no to severe atherosclerosis of the epicardial coronary arteries (Table). Within the AMI group and control group, the degree of atherosclerosis of epicardial coronary arteries did not correlate significantly with the CML immunohistochemical score (not shown).
In AMI patients, CML depositions were clearly present in the small intramyocardial blood vessels (Figure 1B). In these blood vessels, CML adducts were localized in endothelial cells as well as smooth muscle cells. It is noteworthy that these small intramyocardial blood vessels did not show obvious atherosclerotic changes whereas atherosclerosis of epicardial coronary arteries did occur, with lumen obstruction in majority of 50% to 75% (Table). In controls, lumen obstruction of coronary arteries in the majority was 25% to 50%. As to be expected, angina pectoris was significantly more present in AMI patients, compared with controls (Table). In the epicardial coronary arteries, the area of CML positivity of the intima showed a trend to an enhanced CML content in AMI patients (as compared with controls, but the difference was not statistically significant (43.4±7.9 versus 25.6±6.6 AU/mm2; P=0.17).
In contrast, in the intramyocardial blood vessels, the immunohistochemical score per cm2 (defined in Methods) of CML was significantly higher (P<0.002) in AMI patients than in control patients (Figure 1C). We found no significant differences in immunohistochemical score per cm2 for CML between the different infarct durations (Figure 2A). However, the immunohistochemical score per cm2 for CML was significantly higher in all groups of different infarct duration than in control patients (P<0.02 for 0 to 1 day; P<0.01 for 1 to 5 days; P<0.02 for 5 to 14 days). The majority of CML-positive vessels in all groups of different infarct duration were intensely positive and received a maximal “3-score” (Figure 2B). The immunohistochemical score per cm2 for strong CML-positive “3-score” vessels was significantly higher in all groups of different infarct duration than in control patients (P<0.005 for 0 to 1 day; P<0.005 for 1 to 5 days; P<0.04 for 5 to 14 days).
To study if CML was limited to the infarcted area of the heart of patients with AMI, we also stained noninfarcted parts of the heart. Remarkably, no differences in the intensity of CML staining of the small intramyocardial blood vessels were found between infarcted and noninfarcted areas (not shown).
To analyze if the CML adducts were specific for the heart, or were also present in other organs of the same patient, we analyzed CML depositions in small arteries of kidney and lungs of all patients included in this study. In contrast to the heart, no significant difference in CML adducts in small arteries in the lungs and kidneys were found between patients with or without AMI (not shown).
Age, male/female distribution, or smoking did not significantly differ between the control group and AMI group (Table).
Accumulation of CML Is Not Accompanied by the Presence of Myeloperoxidase
MPO can play a role in the formation of CML.14 MPO can be derived from neutrophils but can also be produced by blood vessels themselves. MPO positivity was therefore evaluated in the hearts of AMI patients and healthy controls and compared with CML accumulation. The presence of MPO was limited to a small part within the vessel wall, especially endothelial cells, and was found only in a minority (<5%) of the blood vessels that stained positive for CML (not shown).
Accumulation of CML Is Accompanied by the Presence of E-selectin
To assess whether CML was predominantly associated with activated endothelium we analyzed E-selectin expression in the hearts of AMI patients and healthy controls and compared it with CML accumulation. Within the infarcted myocardium, we found a significant increase (P<0.001) of E-selectin positivity in intramyocardial blood vessels compared with controls (Figure 3C). E-selectin was present in the vessel wall, predominantly on endothelial cells, and was found in almost all of the blood vessels that stained positive for CML (Figure 3A, 3B), demonstrating colocalization of CML and E-selectin.
AMI in Rats Does Not Cause CML Generation Within 24 Hours of Reperfusion
To investigate whether the formation of CML in the heart was directly related to the occurrence of AMI, we used a rat model of AMI. Heart tissues of these rats were evaluated at different time points after 30 minutes of acute ischemia and subsequent perfusion, which varied from 2 to 24 hours. No CML was detectable in these hearts (Figure 4A), although neutrophils were present, indicative for AMI.
Subsequently, we verified whether the CML antibody used did recognize CML in rats. To that end, we studied peritoneal biopsies of rats that had undergone peritoneal dialysis for 5 weeks. Significant amounts of CML were detected in the peritoneum of these rats (Figure 4B). These data thus demonstrate that CML can be formed in the rat and that the CML antibody is suitable for studies in rats.
Together these findings strongly suggest that the CML adducts as found in small intramyocardial blood vessels in the hearts of patients that have died of AMI were present in advance of the AMI.
Oxidative Stress but not Metabolic Inhibition Causes CML Generation by Endothelial Cells
The CML staining was most striking in the endothelium of the intramyocardial blood vessels of AMI patients. To evaluate whether ischemia by itself can cause CML formation in endothelial cells, we treated human endothelial cells (EC-RF24) with conditions of either ischemia (metabolic inhibition) or oxidative stress (H2O2), followed by reperfusion.
Western blot analysis showed no detectable production of CML residues as a result of ischemia. However, in cells incubated with H2O2, minor accumulations of CML adducts were found (Figure 5). Similar results were obtained with quantification of CML in EC-RF24 lysates using LC-MSMS. In control cells 65 nmol CML/mmol lysine was found, whereas in metabolically inhibited/reperfused cells 78 nmol CML/mmol lysine was found and in cells incubated with H2O2 313 nmol CML/mmol lysine was found.
CML Accumulates in Intramyocardial Blood Vessels of Myocarditis Patients
To investigate whether inflammation in the heart in general induces CML upregulation in intramyocardial blood vessels, we investigated the presence of CML in patients with myocarditis. In these patients, CML depositions were present in the small intramyocardial blood vessels, in particular on the endothelium. CML depositions were significantly increased (P<0.0001) compared with control hearts (Figure 1C). The immunohistochemical score of CML in myocarditis patients in fact was significantly higher than in AMI patients (240.5±52.4 and 49.6±7.3, respectively; P<0.0001).
AMI Followed by 5 Days of Reperfusion in Rats Does Cause CML Generation in Intramyocardial Blood Vessels.
Because myocarditis in humans is accompanied by CML depositions in intramyocardial blood vessels, indicating that chronic inflammation cause CML depositions, we next studied in our rat AMI model whether AMI in the chronic state was capable of inducing CML depositions. Therefore, rats were subjected to acute myocardial ischemia and subsequent perfusion for 5 days. At 5 days of infarction, granulation tissue including lymphocytes and macrophages were detected in the heart indicative for chronic inflammation (not shown).
Although minor background staining was found in cardiomyocytes, a clear positive CML signal was found especially in endothelial cells, but also in smooth muscle cells of intramyocardial blood vessels in infarction and noninfarction areas (Figure 4C). Therefore, in rat hearts after AMI, CML depositions can be found in blood vessels, but only in the chronic phase.
In the present study we found CML depositions in small intramyocardial blood vessels in the heart of patients with AMI, irrespective of the age of infarction and the degree of atherosclerosis of epicardial coronary arteries. CML was found in infarcted and noninfarcted areas of the heart.
AMI in general is related to the process of atherosclerosis of epicardial coronary arteries and its complications, such as thrombosis, bleeding within atherosclerotic plaques, and particularly plaque rupture. The questions then raised are how CML is formed, and what is the function of these depositions? It is known that formation of CML may proceed by multiple routes, namely glycation followed by an oxidative cleavage of Amadori adducts,12,25 auto-oxidative glycosylation,13,26 reaction of proteins with nonglucose carbohydrates11 and/or via lipoxidation.14 Next, CML can be formed via the reaction of proteins with products of MPO.14 However, in the small nonatherosclerotic intramyocardial blood vessels, the number of vessels positive for CML, and the intensity of this staining were considerably higher than for MPO. We cannot exclude that MPO activity in these intramyocardial blood vessels has been transient and disappeared again, but the fact that in all cases only a minority of the vessels was positive for MPO makes it unlikely that MPO is playing a major role in CML formation.
CML also has been hypothesized to represent a marker for chronic hypoxic stress.16 In patients with AMI, a phase of (acute) hypoxia is induced. However, we found no differences in CML accumulation in the infarcted area of the heart, and control noninfarcted areas in the heart of the same patient. Although an episode of global ischemia of the whole heart in patients with AMI is supposed, it is difficult to explain CML positivity as a result of ischemia alone. Furthermore, we could not detect CML depositions in intramyocardial blood vessels in rats with AMI, including episodes of reperfusion up to 24 hours. This indicated that CML is not induced in the heart by ischemia/reperfusion in the acute phase. In line with this, also in isolated endothelial cells, ischemia and/or reperfusion did not result in CML formation. This makes it unlikely that in patients with AMI, CML is formed as a direct result of AMI.
Another explanation for the formation of CML might be chronic inflammation. However, it is unlikely that chronic inflammation of the epicardial coronary arteries is playing a role herein, as we did not find a correlation between the degree of atherosclerosis and CML depositions in control nor in AMI patients. We did not find a significant difference in the CML positivity of these epicardial coronary arteries between AMI patients and controls.
It is known that inflammation plays a role in the formation of CML.27 In line with this, in the present study we found that CML positivity colocalized with E-selectin–positive endothelial cells. In addition we have shown that in non-AMI, nondiabetes patients having myocarditis, CML depositions are present on intramyocardial blood vessels, with even a 5-fold increase compared with AMI patients. In line with this, in the rat, AMI-induced inflammation in the chronic phase induced CML on intramyocardial blood vessels in infarction and noninfarction areas. Remarkably, also in humans we found CML in the noninfarction areas. This therefore may suggest that any local inflammatory response on endothelium is capable of inducing CML depositions on intramyocardial blood vessels. Such an inflammatory response may lead to AMI, because it is known that in patients with fulminant myocarditis, secondary AMI can occur.28
Therefore, the combination of the human and animal studies suggests that CML already is present in intramyocardial blood vessels of the hearts of patients that develop AMI. At this moment, however, we do not know the time frame during which this CML positivity is formed. Nevertheless, this finding may be important, as it has been reported that especially in endothelial cells CML-modified proteins can act as a ligand for RAGE (receptor of AGE),8 thereby causing processes linked to inflammatory complications.29 Furthermore, it is known that patients with diabetes have a higher risk of AMI.30,31 We have shown that the CML accumulates in heart tissue in patients with diabetes.17
Whether the accumulation of CML in small intramyocardial blood vessels of the heart indeed does contribute to the induction of AMI or merely reflects the occurrence of an AMI-inducing process remains uncertain. Validation of CML as a risk indicator for AMI will be the subject of a further prospective study, in which CML levels in the blood will be analyzed in patients with stable AP, unstable AP, and AMI.
Sources of Funding
Dr Niessen is a recipient of the Dr E. Dekker programme of the Netherlands Heart Foundation (D99025). This study was financed by Stichting NUTS-OHRA (SNO-T-04-16). Dr Schalkwijk is supported by a fellowship from the Diabetes Fonds Nederland.
A.B. and P.A.J.K. contributed equally to this study.
Original received March 21, 2006; final version accepted August 17, 2006.
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