Doctor of Philosophy / Food Science Institute / J. Scott Smith / Advanced glycation endproducts (AGEs) are formed in many cooked meat products via Maillard browning reactions. Current research suggests consumption of these compounds may be a contributor to chronic diseases such as diabetes and heart diseases. Thus, information on the prevalence and inhibition of these compounds in food is desirable. The first objective was to determine the AGE content, as determined as N[superscript]ε-carboxymethyllysine (CML) level, in cooked meat and fish prepared by general cooking methods recommended by U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). We found AGE was detected in all the cooked samples, but the levels depended on the different cooking conditions. Broiling and frying at higher cooking temperatures produced higher levels of CML and broiled beef contained the highest CML content (21.84 μg/g). However, the baked salmon (8.59 μg/g) and baked tilapia (9.72 μg/g) contained less CML as compared to the other samples. In order to investigate the inhibitory effect of selected natural antioxidant on AGEs formation in cooked meat, four cereal brans, wheat (Jagger, JA), triticale (Spring Triticale, ST; Thundercale, TH), and Rye (RY) bran were added to beef patties before cooking. RY (42.0% inhibition), ST (27.5% inhibition), and TH (21.4% inhibition) brans significantly decreased CML formation compared with the control. The inhibition of CML was correlated to the water-holding activity (WHC) of the samples, and the radical scavenging activity of the brans. The effect of cereal bran extracts (JA, ST, TH, and RY), was studied in a bovine serum albumin and glucose (BSA-GLU) model system. The ST extract significantly (P <0.05) inhibited CML formation compared to the control group. ST particularly contained vanillic acid (VA), chlorogenic acid (CHA), gentisic acid (GEA), and ferulic acid (FA), where GEA and CHA mitigated CML with an average percentage decrease of 29.6% for CHA and 51.1% for GEA. It therefore may be useful in preventing AGEs formation by using ST bran as a food addictive, which contains abundant phenolic acids. In summary, current dietary AGEs database will provide important information for use in estimating AGEs exposure, and also these data demonstrate that a significantly reduced intake of dietary AGEs can be achieved by low heat AGE cooking methods such as baking, which can be used at home or in the meat industry. Cereal bran addition to meat products may reduce formation of AGEs that is a desired attribute for the processed meat products industry.

Methylglyoxal (MG), a metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation end products (AGEs). Increased MG production, which in turn gives rise to AGEs, has been linked to the development of complications in diabetes. However, the role of MG and AGEs in hypertension has not been investigated widely. The previous study from our laboratory showed that the cellular levels of MG and MG-induced AGE formation are significantly higher in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) than those from normotensive Wistar-Kyoto rats (WKY). Using immunofluorescence staining with specific monoclonal antibodies against MG-induced AGEs, the present studies show a strong association of MG and its AGE products (Nå-carboxyethyl-lysine and Nå-carboxymethyl-lysine) with hypertension in SHR. The blood pressure of SHR was not different from that of WKY rats at 5 wks of age. From 8 wks onwards, blood pressure was significantly elevated compared to age-matched WKY rats. Importantly, this increase in blood pressure coincided with an elevated MG level in plasma and aorta of SHR in an age-dependent fashion compared to age-matched WKY rats, although no difference was observed in blood glucose levels between these two strains. Our data showed an increased MG level in plasma and aorta, but not in kidney or heart, in SHR at an early age of 8 wks, suggesting, in addition to diabetes/hyperglycemic or hyperlipidemic conditions, the accumulation of MG in blood vessel walls plays an important role in the development of hypertension or its complications even in the absence of diabetes. Moreover, we observed increased blood pressure and vascular remodeling in Sprague Dawley rats which had been treated to increase endogenous MG and related AGEs. After inhibiting MG and MG-induced AGE generation in SHR, hypertension development in this genetic hypertension model was delayed and vascular remodeling was reversed. Our data indicate that increased MG and AGE formation may play an important role in the development of hypertension.

Methylglyoxal (MG), a metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation end products (AGEs). Increased MG production, which in turn gives rise to AGEs, has been linked to the development of complications in diabetes. However, the role of MG and AGEs in hypertension has not been investigated widely. The previous study from our laboratory showed that the cellular levels of MG and MG-induced AGE formation are significantly higher in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) than those from normotensive Wistar-Kyoto rats (WKY). Using immunofluorescence staining with specific monoclonal antibodies against MG-induced AGEs, the present studies show a strong association of MG and its AGE products (Nå-carboxyethyl-lysine and Nå-carboxymethyl-lysine) with hypertension in SHR. The blood pressure of SHR was not different from that of WKY rats at 5 wks of age. From 8 wks onwards, blood pressure was significantly elevated compared to age-matched WKY rats. Importantly, this increase in blood pressure coincided with an elevated MG level in plasma and aorta of SHR in an age-dependent fashion compared to age-matched WKY rats, although no difference was observed in blood glucose levels between these two strains. Our data showed an increased MG level in plasma and aorta, but not in kidney or heart, in SHR at an early age of 8 wks, suggesting, in addition to diabetes/hyperglycemic or hyperlipidemic conditions, the accumulation of MG in blood vessel walls plays an important role in the development of hypertension or its complications even in the absence of diabetes. Moreover, we observed increased blood pressure and vascular remodeling in Sprague Dawley rats which had been treated to increase endogenous MG and related AGEs. After inhibiting MG and MG-induced AGE generation in SHR, hypertension development in this genetic hypertension model was delayed and vascular remodeling was reversed. Our data indicate that increased MG and AGE formation may play an important role in the development of hypertension.

<p>Introduction: Diabetes mellitus is a major risk factor for tendon pain, injury, and pathology. Surprisingly, tendon problems persist in diabetic patients with superior blood glucose control (HbA1c<6.5), suggesting that alternative mechanisms contribute to this problem. Advanced glycation end products (AGEs) have been implicated in several diabetes-related complications, but their role in diabetic tendon pathology has not fully been explored. To expand our understanding of AGE-mediated tendon pathology, the following five studies were completed.</p><p>Study 1: Streptozotocin-induced diabetes alters transcription of multiple genes necessary for extracellular matrix remodeling in rat patellar tendon. RNA was isolated from the patellar tendon of non-diabetic (control, n=9), 1-week diabetic (acute, n=8), 10-weeks diabetic (chronic, n=7), and insulin treated 10-weeks diabetic (insulin, n=8) rats. Determination of mRNA transcripts was completed using droplet digital PCR (ddPCR). Our findings indicated that STZ-induced diabetes results in rapid and large changes in the expression of several genes that are key to extracellular matrix (ECM) remodeling, maintenance, and maturation.</p><p>Study 2: Advanced glycation end products suppress mitochondrial function and proliferative capacity of Achilles tendon-derived fibroblasts. Using an <i>in vitro</i> cell culture system, rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (0, 50, 100, and 200μg/ml) for 48 hours in normal glucose (5.5mM) and high glucose (25mM) conditions. Our findings demonstrate that tendon fibroblasts treated with AGEs display reduced ATP production, electron transport efficiency, and proliferative capacity. These impairments were coupled with alterations in mitochondrial DNA content and expression of genes associated with ECM remodeling, mitochondrial energy metabolism, and apoptosis.</p><p>Study 3: Descriptive transcriptome analysis of tendon derived fibroblasts following <i>in vitro</i> exposure to advanced glycation end products. Rat Achilles tendon fibroblasts were treated with glycolaldehyde-derived AGEs (200μg/ml) for 48 hours in normal glucose (5.5mM) conditions. Total RNA was isolated and the PolyA⁺library was sequenced. We demonstrate that tendon fibroblasts treated with 200μg/ml of AGEs differentially express 2,159 gene targets compared to fibroblasts treated with an equal amount of bovine serum albumin (BSA)-Control. Our findings suggest that AGEs disrupt the tendon fibroblast transcriptome on a large scale and that these pathways may contribute to the development and progression of diabetic tendinopathy.</p><p>Study 4: Evaluation of tendon healing in a mouse model of elevated serum advanced glycation end products following tendon injury. Mice received daily BSA-Control or AGE-BSA injections (200μg/ml) for two weeks prior to creation of a tendon injury in the central third of both patellar tendons. Animals assigned to an exercise group began a moderate treadmill protocol (13 meters/min, five days/week, five weeks) one week following injury and all animals continued to receive injections until termination. We demonstrated that based on our injection dose and schedule, that serum AGEs are significantly elevated to ~200μg/ml, levels that are typically seen in type II diabetic patients. Additionally, a main effect for AGEs was observed in genes related to cell proliferation (Mybl2), mitochondrial function (Bcs1l), and growth factors (Fgf2). However, moderate treadmill exercise did not alter gene markers, such as Ctgf and Fgf2, which are makers of a tendon healing response. Our findings suggest that AGEs modulate tendon gene expression following patellar tendon injury, with no effect of moderate treadmill exercise.</p><p>Study 5: Serum levels of advanced glycation end products and their relationship to patellar tendon properties in diabetes. Subjects (n=32) from a full spectrum of diabetes status, including no history of diabetes were recruited for a cross-sectional study. A fasted blood sample was collected and magnetic resonance imaging (MRI) of the knee was completed. Both current HbA1c and previous diagnosis was used to stratify collected data. Additionally, a full correlation matrix of all measured variables was created to establish relationships that could be used to predict tendon pathology in diabetes. Our findings demonstrate that diabetes is associated with smaller patellar tendon dimensions, which is in disagreement with the literature. Further, we show that changes to body weight normalized (BWN) tendon cross-sectional area (CSA) occur independent of circulating N^ε-(carboxymethyl)lysine (CML) levels. These new data suggest that alternative mechanisms contributing to tendon pathology in diabetes deserve attention.</p>

In der vorliegenden Arbeit wurde aneurysmatisches Aortengewebe von Patienten mit bikuspider oder trikuspider Aortenklappe untersucht. Im Laufe des Lebens ist die bikuspide Aortenklappe als häufigste angeborene Anomalie des Herzens mit zahlreichen, potentiell lebensbedrohlichen Komplikationen verbunden. Betroffene Patienten zeigen eine frühere Entwicklung und rapidere Progression von Dilatationen und – im schlimmsten Fall – Dissektionen der Aorta ascendens. Die Ätiologie dessen konnte bis dato nicht ausreichend geklärt werden. Hintergrund der Studie war eine Untersuchung von Branchetti et al., wobei eine Erhöhung von RAGE im Plasma bei Patienten mit bikuspider Klappe nachgewiesen werden konnte. Daraus wurde die Hypothese entwickelt, dass eine Expressionserhöhung von RAGE und dessen Liganden AGE im Aortengewebe selbst ursächlich mit der Aortendilatation verbunden sein könnte. In Proben von 93 Patienten wurde mittels Western Blot, ELISA und Immunhistochemie die Expression von RAGE und AGE untersucht. Hierbei zeigte sich eine signifikante Expressionserhöhung beider Proteine im Aortenaneurysma bei bikuspider Klappe im Vergleich zu Patienten mit trikuspider Aortenklappe. Auch die exemplarisch angefertigten Immunhistologien stützen diese Ergebnisse. Mögliche Folgen können Steifigkeitserhöhung der Aortenwand, Aktivierung von Matrixmetalloproteinasen sowie Erhöhung des oxidativen Stresses sein. Neben der Expression im aneurysmatischen Aortengewebe wurden auch Plasmaproben hinsichtlich AGE und RAGE analysiert, wobei sich keine Erhöhung feststellen ließ. Die Ergebnisse der Studie, die eine RAGE – Erhöhung im Plasma detektierten und ihn somit als potentiellen Biomarker für eine bikuspide Klappe diskutierten, ließen sich bei der vorliegenden Untersuchung einer kleineren Stichprobe nicht bestätigen. Ebenso stellt sich die Etablierung eines Biomarkers als anspruchsvolle Aufgabe dar. Eine Eignung von RAGE als Biomarker zur Identifikation von Patienten mit bikuspider Klappe ist kritisch zu betrachten.:Inhaltsverzeichnis Bibliographische Beschreibung Abkürzungsverzeichnis 1. Einleitung 1.1. Die bikuspide Aortenklappe (BAV) 1.1.1. Prävalenz 1.1.2. Klassifikation 1.1.3. Ätiologie 1.1.4. Assoziierte Pathologien 1.1.5. Hypothesen der Dilatationsentstehung 1.1.6. Diagnostik 1.1.7. Therapie 1.2. RAGE und AGE 1.2.1. Advanced Glycation End Products (AGE) 1.2.2. Receptor for Advanced Glycation End Products (RAGE) 1.2.3. Interaktion von AGE und RAGE 1.2.4. Bezug zum thorakalen Aortenaneurysma 2. Zielstellung 3. Material 3.1. Allgemeine Geräte 3.2. Allgemeine Materialien 3.3. Allgemeine Chemikalien 3.4. Proteinextraktion 3.5. Proteinkonzentrationsbestimmung 3.6. SDS – Gelelektrophorese 3.7. Antikörper (AK) 3.8. Western Blot Analyse 3.9. Enzyme – linked Immunosorbent Assay (ELISA) 3.10. Immunhistochemie (IHC) 3.11. Software 4. Methoden 4.1. Patientenpopulation und Probengewinnung 4.2. Isolation der Proteine aus Aortengewebe 4.3. Konzentrationsbestimmung nach BCA – Methode 4.4 Elektrophoretische Auftrennung der Proteine 4.5. Detektion von AGE und RAGE mittels Western Blot Analyse 4.6. Nachweis von AGE und RAGE mittels ELISA 4.7. Immunhistochemische Färbung von AGE und RAGE 4.8. Statistische Auswertung 5. Ergebnisse 5.1. Patientenpopulation 5.2. Expression von AGE in humanen aneurysmatischen Gewebeproben der Aorta ascendens 5.2.1. Analyse der AGE – Expression mittels Western Blot 5.2.2. Analyse der AGE – Expression mittels ELISA 5.2.3. Darstellung der Lokalisation von AGE in der Aortenwand mittels Immunhistochemie 5.3. Expression von RAGE im Aortengewebe 5.3.1. Analyse der Expression von RAGE mittels Western Blot 5.3.2. Analyse der RAGE – Expression mittels ELISA 5.3.3. Darstellung der Lokalisation von RAGE in der Aortenwand mittels Immunhistochemie 5.4. Bestimmung der Plasmaspiegel von AGE und RAGE in ausgewählten Plasmaproben 6. Diskussion 6.1. Expressionserhöhung von AGE in der Aortenwand von Patienten mit BAV 6.1.1. Mögliche Ursachen der Expressionserhöhung 6.1.2. Zusammenhang von AGE und Gefäßsteifigkeit 6.2. Expressionserhöhung von RAGE im Aortengewebe von Patienten mit BAV 6.2.1. Ätiologie der Expressionserhöhung unter Einbeziehung der Liganden 6.2.2. Folgen der RAGE – Erhöhung und ihr Einfluss auf die Gefäßwand 6.3. RAGE in seiner Rolle als Biomarker Schlussfolgerung Limitationen 7. Zusammenfassung 8. Literaturverzeichnis 9. Abbildungsverzeichnis 10. Tabellenverzeichnis Erklärung über die eigenständige Abfassung der Arbeit Lebenslauf Danksagung

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

Over the last few decades, the importance of inflammation in the initiation and perpetuation of cardiovascular (CV) disease has become increasingly recognized. Patients with Rheumatoid Arthritis (RA) have been shown to have an increased risk of premature death, occurring predominantly due to an increased rate of premature CV disease. The presence of an adverse risk factor profile in RA is well established, but does not fully explain the excess risk. It is clear that chronic inflammation is a major pathogenic mechanism in atherosclerosis, and this is likely to explain at least some of the increased risk of CV disease in subjects with RA. Carotid intima-media thickness (cIMT) measured by ultrasound, is a good non-invasive approach to measurement of atherosclerotic burden, and is increased in preclinical atherosclerotic disease. cIMT is significantly higher in patients with RA than age and sex matched controls. The Receptor for Advanced Glycation End Products (RAGE) may be important for the perpetuation of chronic inflammation. This cell surface receptor molecule is upregulated at sites of chronic vascular inflammation, and can be signalled by a range of proinflammatory ligands as well as advanced glycation end-products. The gene has a number of polymorphisms, and the Glycine 82 Serine polymorphism has a prevalence of about 10% in Caucasians. Patients with RA are more likely to have this polymorphism than control subjects, as the gene is in linkage disequilibrium with DRB1*0401, one of the RA susceptibility alleles. There is evidence that ligation of RAGE in monocytes derived from donors with the Ser 82 allele signals an enhanced NF-kB and p38 MAP Kinase cellular response, associated with production of pro-inflammatory cytokines. In this study, I hypothesized an association between the RAGE 82 Ser polymorphism of this receptor, which is enriched in RA, and the risk of CV disease in subjects with RA. To investigate whether RAGE 82Ser is associated with CV disease in RA, I examined events, risk factors, features of RA and RAGE 82Ser, in 232 patients with RA attending a tertiary referral hospital. Carotid intima-media thickness was measured using carotid duplex scanning in 137 of those patients. CV events, duration and severity of RA, and CV disease risk factors were determined using patient questionnaires, chart review, laboratory analysis, and radiographs. DNA was typed for HLA-DRB1 genes and the RAGE 82Ser polymorphism. Twenty percent of patients carried the RAGE 82Ser allele. More than 20% of the cohort had suffered a vascular event. Increasing age, elevated fasting glucose, a history of hypercholesterolemia, and a shorter duration of RA were significantly associated with events. RAGE 82Ser was protective against CV events in this cohort. RA patients with RAGE 82Ser had lower LDL levels and LDL/HDL ratio. cIMT was independently and significantly associated with increasing age, male sex, hypertension, low BMI, and the number of pack years of smoking, but not RAGE genotype. Multiple factors, both CV and RA disease-related, contribute to atherosclerosis in established RA. These data suggest RAGE genotype may contribute to the risk of CV events in RA. The role of RAGE genotype requires further study in inception cohorts examining CV events to better understand its contribution to RA-associated CV disease.

Advanced glycation end products (AGEs) are molecules formed through the nonenzymaticglycation of proteins and are central to the development of cardiovascularcomplications of diabetes including heart failure. AGEs influence cellular function throughthe cross-linking of cellular proteins as well as through actions on cell surface receptors,the most common of which is (RAGE). However, it is still unclear whether AGEs contributeto myocardial abnormalities observed in diabetes through direct myocardial actionsmediated through the RAGE receptor and if so, their underlying mechanisms of action. Wehave therefore investigated the effects of AGEs on calcium handling in isolated adultmouse cardiomyocytes and cultured neonatal rat cardiomyocytes (NRCM) andcharacterised their underlying mechanisms of action in NRCM.Standard molecular techniques were used. Western blot showed expression of RAGEreceptor in mouse whole heart tissue and in both NRCM and adult mouse cardiomyocytes. Incubation of NRCM for 24 hours with AGEs showed a dose dependant reduction ofcalcium transient amplitude with a maximum of 50% at 1 g/l (P<0.01) accompanied with32% reduction in SR calcium content with no detectable changes in calcium handlingproteins expression. We demonstrated a 24% increase (P<0.01) in the production ofreactive oxygen species (ROS) in AGE treated cardiomyocytes induced by enhancedNADPH oxidase activity (P<0.05) with subsequent activation and translocation of NF-kB, atranscriptional factor from the cytoplasm to the nucleus. Activation of NF-kB induced a56% increase in iNOS gene protein expression (P<0.01), a downstream target of NF-kBwhich was accompanied by a significant increase in NO production (P<0.05). Wedemonstrated nitrosylation of several key cellular proteins involved in excitationcontractioncoupling including the Ryanodine receptor and SERCA2a as detected byimmunofluorescence. In conclusion, our work provides insights into novel pathophysiological mechanisms thatunderlie the development of heart failure in diabetes. We demonstrate the presence andfunctionality of AGE receptors in myocardium and show that AGEs inhibit excitationcontractioncoupling through increased ROS production leading to activation andtranslocation of NF-kB from the cytoplasm to the nucleus resulting in increase in NOproduction. Concomitant increases in intracellular levels of ROS and NO favours theproduction of peroxynitrite with subsequent nitrosylation of key cellular proteins involved inthe process of excitation-contraction coupling such as the Ryanodine receptor andSERCA2a. This study provides novel insights into the role of AGEs in inducing myocardialdamage in diabetes mediated through RAGE receptor and independent from the vascular effects.

Expression of the Receptor for Advanced Glycation End Products (RAGE) and is upregulated in a several cancers. Based on published studies, we hypothesized that RAGE, when overexpressed in pancreatic cancer cells, will promote cell proliferation and migration. To study the role of RAGE in pancreatic cancer, we selected the human pancreatic cancer cell-line PANC-1, and stably transfected the cells with full length RAGE to generate model cell-lines that overexpress RAGE. We obtained two cell-lines PANC-1 FLR2 and PANC-1 FLR3 and examined the influence of RAGE on cellular properties. A significant increase in proliferation but a reduction in migratory abilities of PANC-1 FLR2 and PANC-1 FLR3 cells was observed. The increase in proliferation and reduction in migration was reverted upon knockdown of RAGE in PANC-1 FLR2 cells with siRNA specific for RAGE. The reduction in migration was supported by the reduced levels of vimentin and several integrins in RAGE transfected cells. Furthermore, we observed a downregulation in FAK, AKT, ERK1/2 and NF-κB activity. Growing evidence supports that RAGE is essential for pancreatic cancer progression. It has also been shown that RAGE facilitates pancreatic tumor cell survival by enhancing autophagy and inhibiting apoptosis. The goal of our study was to determine the effect of RAGE inhibition during gemcitabine chemotherapy on the growth of pancreatic tumor. Hence, we investigated the effect of RAGE inhibitors and their combination with gemcitabine in an orthotopic mouse model of pancreatic cancer using mouse pancreatic cancer cell-line KPC 5508. We used two RAGE inhibitors, an anti-RAGE monoclonal antibody (IgG2A11) and a small molecule RAGE inhibitor (FPS-ZM1). We observed a significant reduction in tumor weights of the mice treated with the combination of IgG2A11 and gemcitabine as compared to gemcitabine alone treated mice. The reduction in tumor growth was accompanied with increase in p62 levels (marker of autophagy) and increase in levels of cleaved PARP (marker of apoptosis). We also observed reduction in HMGB1 and phosphorylation levels of ERK1/2 in tumors from the group treated with the combination as compared to the gemcitabine alone treated group. / North Dakota State University. College of Health Professions / NIH Grant # P20 GM109024 from the National Institute of General Medicine