Advanced Glycation End Product (AGE) and Soluble Receptor of AGE (sRAGE) Levels in Relation to Periodontitis Severity and as Putative 3-Year Outcome Predictors in Patients Undergoing Coronary Artery Bypass Grafting (CABG).

Tissue concentrations of advanced glycation end product (AGE) and peripheral soluble receptor of AGE (sRAGE) levels may be associated with periodontitis severity. Both parameters and periodontitis might serve as outcome predictors for patients undergoing coronary artery bypass grafting (CABG). This study aimed to investigate possible associations between periodontitis and AGE/sRAGE. Ultimately, we wanted to examine whether AGE, sRAGE, and severe periodontitis are associated with the incidence of new cardiovascular events within 3 years of follow-up after CABG. Ninety-five patients with coronary vascular disease (CVD) (age 69 years, 88.3% males) needing CABG surgery were included. Periodontal diagnosis was made according to the guidelines of the "Centers for Disease Control and Prevention (CDC)" (2007) and staged according to the new classification of periodontal diseases (2018). AGE tissue concentrations were assessed as skin autofluorescence (sAF). sRAGE levels were determined by using a commercially available enzyme-linked immunoabsorbance assay (ELISA) kit. Univariate and multivariate baseline and survival analyses were carried out with Mann-Whitney U test, Chi² test, Kaplan-Meier curves with Log-Rank test, and logistic and Cox regression. sAF was identified as an independent risk indicator for severe periodontitis with respect to the cofactors age, gender, plaque index, and diabetes (adjusted odds ratio [OR] = 2.9, p = 0.028). The degree of subgingival inflammation assessed as a percentage of sites with bleeding on probing (BOP) was inversely correlated with sRAGE concentration (r = -0.189, p = 0.034). Both sAF (Hazard Ratio [HR] = 2.4, p = 0.004) and sRAGE (HR = 1.9, p = 0.031) increased the crude risk for new adverse events after CABG. The occurrence of severe periodontitis trends towards a higher risk for new cardiovascular events (HR = 1.8, p = 0.115). Applying multivariate Cox regression, only peripheral arterial disease (adjusted HR = 2.7, p = 0.006) and history of myocardial infarction (adjusted HR = 2.8, p = 0.010) proved to be independent risk factors for cardiovascular outcome. We conclude that sAF may represent a new, independent risk indicator for severe periodontitis. In contrast, sAF, sRAGE, and severe periodontitis were not independent prognostic factors for postoperative outcome in patients undergoing CABG.

Accumulation of glycated proteins suggesting premature ageing in lamin B receptor deficient mice.

Accumulation of advanced glycation end products (AGEs) is accompanied by increased free radical activity which contributes to ageing and the development or worsening of degenerative diseases. Apart from other physiological factors, AGEs are also an important biomarker for premature ageing. Here we report protein modifications (glycation) in a mouse model of lamin B receptor deficient ic J /ic J mice displaying skin defects similar to those of classical progeria. Therefore, we analysed AGE-modifications in protein extracts from various tissues of ic J /ic J mice. Our results demonstrated that pentosidine as well as argpyrimidine were increased in ic J /ic J mice indicating a modification specific increase in biomarkers of ageing, especially derived from glycolysis dependent methylglyoxal. Furthermore, the expression of AGE-preventing enzymes (Glo1, Fn3k) differed between ic J /ic J and control mice. The results indicate that not only lamin A but also the lamin B receptor may be involved in ageing processes.

Soluble form of receptor for advanced glycation end products and incidence of new cardiovascular events among patients with cardiovascular disease.

BACKGROUND AND AIMS: Soluble RAGE (sRAGE) serum level could be a biomarker for atherosclerosis and subsequent diseases such as cardiovascular disease (CVD). Therefore, we wanted to investigate whether peripheral sRAGE level is associated with new cardiovascular events among patients with CVD using the Cox's regression analysis. METHODS: In this three-year longitudinal cohort study, 1002 in-patients with angiographically proven CVD were included. In 933 patients, sRAGE levels were determined by a commercial available ELISA kit at the time of baseline examination. The combined endpoint was defined as myocardial infarction, stroke/TIA (non-fatal, fatal), and cardiovascular death. For risk analysis, sRAGE values were distributed in quartiles. For generation of adjusted hazard ratios (HR), other risk factors for CVD, such as age, gender, current smoking, body mass index, diabetes, hypertension, dyslipoproteinemia, family history of CVD, severe periodontitis, serum levels for C-reactive protein and interleukin-6, were recorded. RESULTS: 886 patients completed the 3-year follow-up. The overall incidence of the combined endpoint was 16%. Patients with sRAGE levels >838.19 pg/ml (fourth quartile) had the highest incidence of recurrent CVD events (24.9% versus 13.1%, p < 0.0001). In multivariate Cox regression with respect to further confounders for CVD, the association between sRAGE and new CVD events was confirmed (HR = 1.616, 95% CI 1.027-2.544, p = 0.038). CONCLUSIONS: Elevated sRAGE serum level is associated with further adverse events in patients with CVD.

Influence of +1245 A/G MT1A polymorphism on advanced glycation end-products (AGEs) in elderly: effect of zinc supplementation.

Advanced glycation end-products (AGEs) stimulate reactive oxygen species (ROS) generation and represent a risk factor for atherosclerosis, while their formation seems to be prevented by zinc. Metallothioneins (MT), zinc-binding proteins exert an antioxidant function by regulating intracellular zinc availability and protecting cells from ROS damages. +1245 A/G MT1A polymorphism was implicated in type 2 diabetes and in cardiovascular disease development as well as in the modulation of antioxidant response. The purpose of this study was to investigate the influence of +1245 A/G MT1A polymorphism on AGEs and ROS production and to verify the effect of zinc supplementation on plasma AGEs, zinc status parameters and antioxidant enzyme activity in relation to this SNP. One hundred and ten healthy subjects (72 ± 6 years) from the ZincAge study were supplied with zinc aspartate (10 mg/day for 7 weeks) and screened for +1245 MT1A polymorphism. +1245 MT1A G+ (Arginine) genotype showed higher plasma AGEs and ROS production in peripheral blood mononuclear cells (PBMCs) than G- (Lysine) one at the baseline. No significant changes after zinc supplementation were observed for AGEs, ROS and MT levels as well as for enzyme antioxidant activity in relation to the genotype. Among zinc status parameters, major increases were observed for the intracellular labile zinc (iZnL) and the NO-induced release of zinc in PBMCs, in G+ genotype as compared to G- one. In summary, +1245 G+ carriers showed increased plasma AGEs and ROS production in PBMCs at baseline and a higher improvement in iZnL after zinc intervention with respect to G- individuals.

RAGE influences the development of aortic valve stenosis in mice on a high fat diet.

Advanced glycation end product (AGE) accumulations as well as a high fat diet are associated with cardiovascular diseases. AGEs are recognized by several receptor molecules of which the receptor of AGEs (RAGE) is currently the most intensively studied. Activation of RAGE causes an unfavorable pro-inflammatory state. The hypothesis of this study was that metabolic stress due to a high fat diet results in the development of aortic valve stenosis and that knockout of RAGE should be protective. Six week old male C57BL/6N and C57BL/6N RAGE-/- mice (n=28) were randomly assigned to 4 groups and fed with normal or high fat diet for 32weeks. Weight gain was determined weekly. At the start of the experiment and after 2, 4 and 7months, echocardiographic assessments of the aortic valve were made. At the end of the experiment, plasma lipid levels and histological changes of the valves were determined. The high fat diet resulted in accelerated weight gain. However, after 7month, only C57BL/6 mice developed increased trans-aortic-valve velocities, leaflet thickness and reduced valve area index (p<0.0001). Immunohistochemistry of the aortic valves revealed in C57BL/6N mice on a high fat diet more calcification, AGE accumulation and RAGE expression when compared to normal fed control. Hearts and aortic valves of RAGE-/- mice showed less morphometric changes, calcification and AGE accumulation. After 7months of high fat feeding C57BL/6 mice (p<0.0001) as well as RAGE-/- mice (p=0.007) had significantly increased cholesterol levels compared to normal fed control, however RAGE-/- mice were probably protected due to a better HDL/LDL ratio when compared to wild type animals (p=0.003). These data suggest that AGEs and RAGE are involved in the development of obesity, hypercholesterolemia and aortic valve changes due to metabolic stress from high fat intake.

Advanced glycation end product associated skin autofluorescence: a mirror of vascular function?

Advanced glycation end products (AGEs) seem to be involved in aging as well as in the development of cardiovascular diseases. During aging, AGEs accumulate in extracellular matrix proteins like collagen and contribute to vessel stiffness. Whether non-invasive measurement of AGE accumulation in the skin may reflect vessel function and vessel protein modification is unknown. Herein we set out to analyze the AGE-modifications in the collagens extracted from residual bypass graft material, the skin autofluorescence reflecting the accumulation of AGEs in the body as well as the pulse wave velocity reflecting vessel stiffness. Collagen types I and III (pepsin digestible collagen fraction) were isolated from the veins of 52 patients by proteolysis. The residual collagen fraction was further extracted by collagenase digestion. Collagen was quantified by hydroxyproline assay and AGEs by the AGE intrinsic fluorescence. Skin autofluorescence was measured with an autofluorescence reader; pulse wave velocity with the VICORDER. The collagen AGE autofluorescence in patient vein graft material increased with patient age. The pepsin digestible collagen fraction was significantly less modified in comparison to the collagenase digestible fraction. Decreasing amounts of extracted collagenase digestible collagen correspond with increasing AGE autofluorescence. Skin autofluorescence and vessel stiffness were significantly linked to the AGE autofluorescence of the collagenase digestible collagen fraction from graft material. In conclusion we have found that skin autofluorescence and pulse wave velocity as non-invasive parameters significantly correlate with the AGE contained in graft material and therefore are strong predictors of vessel AGE modifications in patients with coronary heart disease. Whether the analysis of the skin autofluorescence leads to an improvement of the risk stratification in patients suffering from cardiovascular disease has to be further tested.

Amyloid-ß oligomers in cerebrospinal fluid are associated with cognitive decline in patients with Alzheimer's disease.

Oligomers of the amyloid-ß peptide (Aß) are thought to be the most toxic form of Aß and are linked to the development of Alzheimer's disease (AD). Here, we used a flow cytometric approach for the detection and assessment of oligomers in cerebrospinal fluid (CSF) from AD patients and other neurological disorders. 30 CSF samples from patients suffering from AD (n = 14), non-demented controls (n = 12), and other neurological disorders (dementia with Lewy bodies, n = 2; vascular dementia, n = 1; primary progressive aphasia, n = 1) were analyzed for the presence of Aß-oligomers by flow cytometry. The CSF levels of total tau (t-tau), phosphorylated tau (p-tau), and amyloid-ß (Aß)42 were determined using ELISA. CSF Aß-oligomer levels in AD patients were elevated in comparison to the non-AD group (p = 0.073). The ratio Aß-oligomers/Aß42 was significantly elevated in AD subjects compared to non-AD subjects (p = 0.001). Most important, there was a negative correlation between the amount of Aß-oligomers and the Mini-Mental Status Exam score (r = -0.65; p = 0.013) in AD patients. The detection of Aß-oligomers using flow cytometry analysis seems to be useful in assessing the stage of AD. This is a novel and important finding as none of the currently used CSF biomarkers are clearly associated with dementia severity.

Insulin-like growth factor binding proteins-2 and -4 enhance the migration of human CD34-/CD133+ hematopoietic stem and progenitor cells.

The insulin-like growth factor (IGF) system is involved in cell migration, which plays an important role in cancer progression. It has been shown that cancer progression correlates with the level of circulating human hematopoietic stem and progenitor cells (HSPCs) expressing CD34 and/or CD133. However, it is unknown whether factors released from cancer cells, including soluble compounds of the IGF system, recruit these HSPCs via enhancing their migration. Our study showed the expression of type I IGF receptor (IGF-IR) in human HSPCs expressing CD34 and/or CD133. In an indirect co-culture model, soluble factors released from human lung epithelial cancer cells (H358, H322) increased the migration of CD34-/CD133+ cells towards cancer cells, whereas migration of CD34+/CD133+ or CD34+/CD133- cells remained unchanged. The lung epithelial cancer cell lines H358 and H322, exhibited a high expression of IGFBP-2, -4 and -6 but not IGF-I and IGFBP-3. Subsequent analyses with those soluble compounds of the IGF system revealed a dose-dependent stimulating effect of the IGFBP-2 and -4 on the migration of CD34-/CD133+ cells. In contrast, IGF-I and IGFBP-3 and -6 did not influence the migration of CD34-/CD133+ cells. Because IGFBPs are involved in cell migration via IGF-dependent and -independent mechanisms, our study indicates that IGFBP-2 and -4, which are expressed in lung epithelial cancer cells, enhance the migration of CD34-/CD133+ HSPCs independent of IGF-I.

A method for the detection of amyloid-beta1-40, amyloid-beta1-42 and amyloid-beta oligomers in blood using magnetic beads in combination with Flow cytometry and its application in the diagnostics of Alzheimer's disease.

A method for simultaneous quantification of amyloid-beta1-40, amyloid-beta1-42 and amyloid-beta oligomers in human plasma is described. The method consists of a combination of immunoprecipitation using specific antibodies against the different forms of amyloid-beta, and immobilization of the immunocomplexes to magnetic beads. Addition of fluorescence-labelled antibodies which recognize the specific antibodies to the amyloid-beta subsets allows the peptide/associates detection in the sample by flow cytometry. The clinical assay performance was tested using blood samples from Alzheimer disease's patients and control donors. A sensitivity of 70% and a specificity of 81% was achieved.

Detection of amyloid-beta oligomers in human cerebrospinal fluid by flow cytometry and fluorescence resonance energy transfer.

The neuropathology of Alzheimer's disease (AD) has been linked recently to non-fibrillar forms of neurotoxic amyloid-beta (Abeta) oligomers of which high levels are observed in the brain of AD patients. This suggests that Abeta oligomers play a key role in the early events of AD, underlining their potential for the early diagnosis of the disease. We have developed an extremely sensitive assay for the detection of oligomeric and fibrillar structures of Abeta that is based on multiparametric analysis of data obtained by flow cytometry and fluorescence resonance energy transfer (Fret). The assay readily detects Abeta oligomers in human cerebrospinal fluid (CSF) as verified by dot blot of the isolated particles. By measuring 174 CSF samples of non-demented control patients with various neurological disorders a high reliability and reproducibility of the method could be demonstrated.