Kathleen Jacobs

Role of advanced glycation end products in cellular signaling

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.

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.

Relationship between cardiac tissue glycation and skin autofluorescence in patients with coronary artery disease

AIM During ageing, advanced glycation end-products (AGEs) accumulate in extracellular matrix proteins like collagen and contribute to a decline in organ function. As skin autofluorescence (sAF) can assess subcutaneous accumulation of fluorescent AGEs, this study aimed to investigate the relationship between AGE-modified cardiac tissue collagen and AGE-related sAF in coronary artery bypass graft (CABG) surgery patients. METHODS Between January 2011 and January 2012, data from 72 consecutive male patients undergoing isolated CABG were prospectively recorded. Collagen fractions were isolated from the right atrial appendages of these patients by proteolysis and collagenase digestion. Collagen was quantified by hydroxyproline assay, and AGEs by AGE-related intrinsic fluorescence; sAF was measured using an autofluorescence reader. RESULTS Biochemical analysis showed that the insoluble cardiac collagen fraction contained the highest amounts of accumulated AGEs; the AGE-related intrinsic fluorescence of this fraction increased with age (P=0.0001), blood glucose (P=0.002), HbA1c (P=0.01) and sAF (P=0.008). CONCLUSION This study demonstrated for the first time a relationship between cardiac tissue glycation and AGE-related sAF. In addition, cardiac tissue glycation was associated with age, blood glucose and long-term glucose values in patients with coronary artery disease.

Novel insights in the dysfunction of human blood-brain barrier after glycation.

The blood-brain barrier (BBB) provides a dynamic and complex interface consisting of endothelial cells, pericytes and astrocytes, which are embedded in a collagen and fibronectin-rich basement membrane. This complex structure restricts the diffusion of small hydrophilic solutes and macromolecules as well as the transmigration of leukocytes into the brain. It has been shown that carbonyl stress followed by the formation of advanced glycation endproducts (AGE=glycation) interfere with the BBB integrity and function. Here, we present data that carbonyl stress induced by methylglyoxal leads to glycation of endothelial cells and the basement membrane, which interferes with the barrier-function and with the expression of RAGE, occludin and ZO-1. Furthermore, methylglyoxal induced carbonyl stress promotes the expression of the pro-inflammatory interleukins IL-6 and IL-8. In summary, this study provides new insights into the relationship between AGE formation by carbonyl stress and brain microvascular endothelial barrier dysfunction.

Advanced Glycation Endproducts Interfere with Adhesion and Neurite Outgrowth

Advanced glycation endproducts (AGEs) represent a non-enzymatic posttranslational protein modification. AGEs are generated by a series of chemical reactions of free reducing monosaccharides, such as glucose, fructose or metabolites of the monosaccharide metabolism with amino groups of proteins. After oxidation, dehydration and condensation, stable AGE-modifications are formed. AGE-modified proteins accumulate in all cells and tissues as a normal feature of ageing and correlate with the glucose concentration in the blood. AGEs are increased in diabetic patients and play a significant role in the pathogenesis of most age-related neural disorders, such as Alzheimer’s disease. We examined the role of AGEs on neurite outgrowth of PC12 cells. We induced the formation of AGEs using the reactive carbonyl compound methylglyoxal (MGO) as a physiological metabolite of glucose. We found that AGE-modification of laminin or collagen interfered with adhesion but not with neurite outgrowth of PC12 cells. Furthermore, the AGE-modification of PC12 cell proteins reduced NGF-induced neurite outgrowth. In conclusion, our data show that AGEs negatively influence neural plasticity.

Glycation of the high affinity NGF-receptor and RAGE leads to reduced ligand affinity.

AGEs are posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between sugars and proteins - a reaction referred to as glycation. Glycation, a feature of ageing, can lead to non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes such as neurodegeneration. In this study we investigated the influence of glycation on the high affinity NGF-receptor TrkA and the AGE-receptor RAGE. We quantified the binding affinity of the TrkA-receptor and RAGE to their ligands by surface plasmon resonance (SPR) and compared these to the binding affinity after glycation. At the same time, we established a glycation procedure using SPR. We found that glycation of TrkA reduced the affinity to NGF by a factor of three, which could be shown to lead to a reduction of NGF-dependent neurite outgrowth in PC12 cells. Glycation of RAGE reduced binding affinity of AGEs by 10-fold.

The skin autofluorescence reflects the posttranslational glycation grade of the matrix protein collagen

Advanced glycation end products (AGEs) seem to be involved in ageing as well as in the development of cardiovascular diseases. Accumulation of AGEs contribute to tissue stiffness and organ dysfunction by crosslinking extracellular matrix proteins like collagen. We aimed to assess whether AGE-modified cardiac tissue collagen and AGE related skin autofluorescence may reflect the cardiac function and have a prognostic value for the outcome of coronary artery bypass surgery patients. Therefore, AGE-modifications in collagen from 72 male patients undergoing isolated coronary artery bypass graft (CABG) surgery were analyzed. Collagen fractions were isolated from the right atrial auricle and the residual bypass graft material (saphenous vein) of these patients and quantified by 4-hydroxyproline assay. AGE modifications were determined by the AGE intrinsic fluorescence (excitation 360nm/emission 440nm). The skin autofluorescence (sAF) as a non-invasive parameter was measured using the AGE reader. The non-extractable collagen contained the highest amounts of AGEs and positively correlates with the patients age (p=0.0001), blood glucose level (p=0.002), HbA1c level (p=0.01) and sAF (p=0.008). The right atrial auricle collagen showed significantly more modifications compared to vein graft material of the same patient (p=0,001). Skin autofluorescence positively correlates with AGE content in cardiac tissue (p=0.01) and therefore could be used as a predictor of tissue stiffness in patients with coronary heart disease.

Dicarbonyls induce senescence of human vascular endothelial cells

RATIONALE Glyoxal (GO) and Methylglyoxal (MGO) are two dicarbonyls involved in the formation of advanced glycation end products (AGEs). Endothelial cells in the vessels are in constant contact with circulating AGEs and dicarbonyls. With this project, we aimed to elucidate the effect of GO and MGO on primary human vascular endothelial cells (HVECs). METHODS Graft material from patients with coronary heart disease was used as HVECs source. HVECs were treated with different concentrations of GO and MGO. β-Galactosidase related senescence activity and cell morphology were analyzed. AGEs as well as p21 protein expression, glyoxalase-I expression and oxidative stress were detected. RESULTS We here provide evidences that GO and MGO induce senescence in primary HVECs. Mechanistically GO and MGO induce senescence by increasing the ROS production, the expression of p21, the accumulation of AGEs and the arrest of HVECs in the G2 cell cycle phase. Aminoguanidine - a dicarbonyl scavenger - abrogated the effect of GO and MGO. CONCLUSION Our data are relevant as they suggest that in diseases with elevated dicarbonyl concentrations, deleterious effects on the endothelium and the development of vascular dysfunction have to be expected. On the other hand, treatment of patients with dicarbonyl scavenger could prevent this.

Analysis of advanced glycation end product modifications in collagen of cardiac tissue and graft material from patients suffering from coronary heart disease

Background: Advanced glycation end products (AGEs) seem to be involved in ageing as well as in the development of cardiovascular diseases. During ageing, the AGEs accumulate in matrix proteins like collagen and fibronectin inducing tissue stiffness. The Bypass operation is the most common intervention in patients suffering from coronary heart disease. However as the majority of the patients are older than 60, the graft material (vein or artery) of these patients is probably enriched on AGE-modifications. To which extent these modifications may affect the quality and functionality of bypass graft material and the heart function after operation is unknown. We analyzed the AGE-modifications in the collagen from the right atrium and residual bypass graft material (saphenous vein and internal thoracic artery) to elucidate in which extension the collagen of graft material is AGE-modified. Methods: Collagen type I and III (soluble fraction) were isolated from veins, arteries and the right atrium by proteolysis with pepsin. The residual insoluble fraction was extracted by digestion with collagenase type I and proteinase K. Collagen was quantified by 4-hydroxyproline assay and AGEs by the AGE intrinsic fluorescence (360nm excitation and 440nm emission). Results: The AGE fluorescence in patient material (vein, atrium) increased with patients age (p=0.0025). Decreasing amounts of extracted insoluble collagen correlate with increased AGE fluorescence (p=0.0001). Comparing vein, artery and atrium of the same patients we isolated equal levels of soluble and insoluble collagen in arteries and atriums, whereas only in veins increased amounts of soluble collagen were extracted in comparison to the insoluble fraction (p<0.05). The soluble fraction in every tissue was significantly less modified in comparison to the insoluble collagen (p<0.05). Conclusion: Ageing is reflected by an increase in AGE related tissue collagen fluorescence. Increase crosslinking of collagen, reflected by high AGE fluorescence, may be the cause of reduced amounts of insoluble extracted collagen. High levels of AGE-modifications are known to block the proteolytic degradation by matrix metalloproteinase. Arteries and cardiac tissue (right atrium) showed significantly more modifications compared to vein graft material of the same patients. Our results may indicate why veins and arteries behave differently as bypass graft material. This will be further evaluated from our group.