Methylglyoxal (MG), a highly reactive dicarbonyl accumulates after myocardial infarction (MI), causing adverse remodelling and cardiac dysfunction. We hypothesized that therapy using bone marrow cells (BMCs) overexpressing glyoxalase1 (Glo1), the main enzyme that metabolizes MG, injected into mouse MI model would translate into better survival of transplanted cells and improve their therapeutic effect. We found that Glo1 expression is significantly reduced at 7 days post-MI. Glo1 BMCs exposed to MG in vitro displayed greater angiogenic potential and reduced reactive oxygen species production compared to wild type (WT) BMCs. However, in the mouse MI model, Glo1 BMCs did not improve cardiac function or vascularity or reduce scar formation compared to WT BMCs and saline treatments. In conclusion, Glo1 overexpression in BMCs does not confer superior therapeutic efficacy for treating MI under the conditions tested.

Myocardial infarction (MI) persists as one of the leading causes of death worldwide. Often patients whom survive the initial injury will develop heart failure characterized by a dilated and functionally incompetent heart. Heart failure (HF) carries a worse prognosis than most cancers, and the only curative therapy to date is heart transplantation. A better understanding of the repair and remodeling processes post-MI, and the development of novel therapies are required to combat this burgeoning medical challenge. This thesis research sought to identify a novel mediator of the impaired cardiac remodeling that often occurs post-MI, and to characterize a biomaterial hydrogel therapy as a novel treatment. We investigated the role of methylglyoxal (MG), an important precursor to advanced glycation end-products (AGE), using a transgenic mouse model to over-express glyoxalase 1 (GLO1). GLO1 is the primary enzyme involved in metabolizing MG and preventing its accumulation. The role for MG and AGEs in MI and HF had been alluded to in the literature, yet no study to date has causally linked them with the loss of function and impaired remodeling of the post-MI heart. We also assessed an injectable hydrogel for the treatment of MI using a mouse model and evaluated the impact of delivery timing on its therapeutic efficacy. In this thesis, we confirmed that MG derived AGEs accumulate post-MI (Chapter 3.1). We show that preventing their accumulation, through GLO1 over-expression, mitigates the loss of function post-MI and positively influences remodeling through reducing final infarct sizes and end-systolic volumes. We demonstrate that this may possibly occur through improving the bone marrow response post-MI by restoring ECM-cell signaling. In Chapter 3.2, we present results of a study assessing the efficacy of a collagen based injectable hydrogel for the treatment of MI, and assessing the role that timing plays into the benefits associated with this therapy by studying 3 separate timepoints including 3 hours, 7 days and 14 days post-MI. We found that the injectable hydrogel preserved cardiac function and reduced infarct sizes. It also positively interacted with the host repair response by reducing chronic inflammation and cell death. The benefits of the therapy depended on when the material was delivered, and we found that the earliest timepoint (3 hours post-MI) proved most beneficial. In Chapter 3.3, we combined the knowledge gained from Chapters 3.1 and 3.2 and functionalized our hydrogel with a flavonoid, Fisetin, that has been shown to scavenge MG and increase the activity of GLO1. We show that this novel functionalized material may be able to restore some function in MI, particularly in settings of low baseline cardiac function. Taken together, the results of this thesis demonstrate that MG accumulates as a result of the ischemia and contributes to the impaired repair resolution and remodeling processes post-MI. This identifies MG as a possible novel target for the treatment of MI. Indeed, we also confirm the role that delivery timing plays into injectable hydrogels post-MI, and present promising results for a functionalized material design to intervene on MG production.

No description available.

Arterial system is a system of vessels distributing blood. Ageing of arterial system leads to two distinct pathologies: atherosclerosis and arteriosclerosis - stiffening of arterial wall. These pathologies can coexist and interfere; however, they differ in their pathogenesis, location, scope and consequences. Progressive loss of elastic properties of large arteries is natural part of vascular ageing. It is directly responsible for several age dependent consequences, such as increase of central systolic pressure or prevalence of isolated systolic hypertension in the elderly. Clinically, central arteries stiffness manifests as aortic pulse wave velocity, which can be quantified, among other methods, using applanation tonometry. There is abundant evidence that aortic pulse wave velocity represents an independent predictor of cardiovascular mortality and morbidity. The most important mechanism in arterial stiffening is repeated mechanical damage which leads to fractures, fragmentation and thinning of elastin. Stiffening of large arteries can be accelerated by several other mechanisms, e.g. deposition of several substances (calcium, advanced glycation end-products, etc.), metabolic turnover of key elements of vascular extracellular matrix (collagen and elastin) or individual genetic susceptibility. In...

L’endothélium est une barrière semi-perméable assurant le maintien de l’homéostasie vasculaire. C’est un régulateur clé du tonus vasculaire, des processus de coagulation et de fibrinolyse, et de l’inflammation. Sa dysfonction est à l’origine de nombreuses pathologies parmi lesquelles figurent les complications vasculaires liées au diabète, notamment les maladies cardiovasculaires, première cause de mortalité chez les patients diabétiques. Le stress oxydant associé aux produits avancés de glycation (AGEs), dont la formation est favorisée par l’hyperglycémie, constitue l’élément central de la dysfonction endothéliale. Ce stress correspond à un déséquilibre entre les défenses antioxydantes et les espèces prooxydantes cellulaires en faveur de ces dernières, et peut être d’origine mitochondriale. L’objectif de mon travail de thèse a été de déterminer les effets des AGEs dérivant de l’albumine (la protéine plasmatique la plus abondante) sur le fonctionnement des mitochondries de cellules endothéliales en culture, en parallèle à une analyse des mécanismes moléculaires impliqués dans le stress oxydant intracellulaire, et des fonctions endothéliales. L’étude mitochondriale s’est principalement axée sur la description des états respiratoires et a révélé des phénotypes associés aux AGEs, variant en fonction du modèle cellulaire endothélial et de la confluence. Par ailleurs, deux autres résultats phares issus de mes investigations correspondent à la mise en évidence de : 1) l’altération des propriétés antioxydantes de l’albumine, ainsi que l’acquisition d’un pouvoir pro-oxydant après glycoxydation ; 2) la contribution des mitochondries au stress oxydant, à travers une communication possible avec la NADPH oxydase, une enzyme produisant des anions superoxydes. Ce travail apporte ainsi un nouvel éclairage sur le déséquilibre redox observé chez les cellules endothéliales dans le cadre de la pathologie diabétique, en relation avec l’aspect mitochondrial. / The endothelium ensures the maintenance of vascular homeostasis. It is a fundamental regulator of vascular tone, coagulation, fibrinolysis, and inflammation. Its dysfunction mediates numerous pathologies, including, among others, diabetes vascular complications, particularly cardiovascular diseases, the major cause of mortality in diabetic patients.Oxidative stress related to advanced glycation end products (AGEs), whose formation is enhanced by hyperglycemia, represents the central element of endothelial dysfunction. This stress is defined as “an imbalance between oxidants and anti-oxidants in favor of the oxidants”, and can originate from mitochondria. The objective of my thesis was to determine the effects of AGEs derived from albumin (the most abundant protein in plasma) on mitochondria of cultured endothelial cells, in parallel to an analysis of the molecular mechanisms involved in intracellular oxidative stress, and endothelial functions.Mitochondrial exploration mainly focused on the description of the respiratory states and revealed AGEs-associated phenotypes, depending on endothelial cell model and cell confluence. Furthermore, two other key results from my investigations correspond to the highlighting of: 1) the alteration of albumin antioxidant properties, as well as the acquisition of a pro-oxidant capacity after glycoxidation processes; 2) the involvement of mitochondria in oxidative stress, through a potential communication with NADPH oxidase, an enzyme that produces superoxide anions.This work brings novel insights into the redox imbalance observed in endothelial cells during diabetes, in relation to the mitochondrial aspect.

Heart failure (HF) induced by aortic pressure over-load is associated with increased coronary artery stiffness. Perivascular adipose tissue (PVAT) and advanced glycation end products (AGE) both promote arterial stiffness. However, the mechanisms by which coronary PVAT promotes arterial stiffness and the efficacy of exercise to prevent coronary stiffness are unknown. The present study hypothesized both chronic continuous and interval exercise training would prevent coronary artery stiffness associated with inhibition of PVAT secreted AGE. Yucatan mininature swine were divided into four groups: control-sedentary (CON), aortic-banded sedentary heart failure (HF), aortic-banded HF continuous exercise trained (HF+CONT), and aortic-banded HF interval exercise trained (HF+IT). Coronary artery stiffness was assessed by ex vivo mechanical testing and coronary artery elastin, collagen and AGE-related proteins were assessed by immunohistochemistry. HF promoted coronary artery stiffness with reduced elastin content and greater AGE accumulation which was prevented by chronic continuous and interval exercise training. HF PVAT secreted higher AGE compared with CON and was prevented in the HF+CONT and HF+IT groups. Young healthy mouse aortas cultured in HF PVAT conditioned media had increased stiffness, lower elastin content and AGE accumulation compared with CON, which was prevented by PVAT from the HF+CONT and HF+IT groups. HF coronary PVAT secreted greater interleukin-6 (IL-6) and IL-8 compared to CON which was prevented by both continuous and interval exercise training regimens. We conclude chronic continuous and interval exercise is a potential therapeutic strategy to prevent coronary artery stiffness via inhibition of PVAT-derived AGE secretion in a pre-clinical mini-swine model of pressure overload-induced HF.

Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity in the world. The risk of CVDs increases with age, tobacco, diabetes, dyslipidemia, obesity and kidney dysfunction. The incidence and prevalence of CVDs demands the development of efficient strategies for prevention and treatment, as well as new biomarkers. The receptor for advanced glycation end-products (RAGE) is implicated in several metabolic and inflammatory disorders. RAGE activation by its multiple ligands, i.e. advanced glycation end-products (AGEs), S100 proteins and amphoterin (HMGB1) induces pro-inflammatory events upon RAGE engagement. The soluble circulating form of RAGE (sRAGE) has been proposed as a biomarker of vascular risk, disease severity and outcome, especially in individuals with diabetes or kidney dysfunction. However, data is controversial since positive and negative correlations are observed for a same disease. Nevertheless, the importance of the ligand-RAGE axis in pathological processes and the wide range of RAGE-binding molecules (from pro-inflammatory proteins to autoantibodies), appreciates the present study.In this thesis, we first investigated effects of RAGE ligands and the recently described anti-sRAGE autoantibodies on sRAGE quantification. We hypothesized that interactions between sRAGE and these molecules could impair sRAGE quantification. On the second part, we evaluated the value of sRAGE and anti-sRAGE autoantibodies as biomarkers of metabolic improvement after bariatric surgery for morbid obesity. Patients were selected from the established cohort ABOS (Lille). RAGE ligands (Nε-carboxymethyllysine, S100A6, S100A12, S100B, HMGB1 and amyloid beta peptide) bind sRAGE at different sites and could potentially impair its quantification through epitope masking. We tested this hypothesis by incubating these ligands, from physiological to pathological concentrations, with recombinant sRAGE and serum to evaluate their effects on sRAGE quantification. Anti-sRAGE autoantibodies were identified and further purified and their effects on sRAGE measurement evaluated. The presence of ligands or anti-sRAGE autoantibodies did not impair recombinant or serum sRAGE quantification. Obesity is a condition of dyslipidemia, glycemia deregulation and inflammation where RAGE is believed to play an important role. We aimed then to investigate the levels of sRAGE and its autoantibodies according to metabolic improvement in obese subjects submitted to weight loss surgery. Patients were highly selected from a well established cohort (morbidly obese patients eligible for gastric bypass, ABOS, Lille). Patients under statins treatment, with kidney dysfunction or hypertension, factors that could affect sRAGE levels, were excluded. In obese patients, significant higher levels of sRAGE and anti-sRAGE autoantibodies were observed before weight-loss surgery. In parallel to body-mass Index, both sRAGE and anti-sRAGE titers were significantly decreased one year after surgery.We demonstrate that the variations of sRAGE levels among the literature are, most likely, not due to an interaction between RAGE ligands and sRAGE. Other hypothesis like the regulation of sRAGE formation and clearance are further discussed. We have, for the first time demonstrated the presence of anti-sRAGE autoantibodies in obese subjects and that their levels decrease after bariatric surgery. Although our data suggest that morbid obese status leads to an autoimmune reactions against sRAGE. Together, our findings argue against sRAGE as a good biomarker but suggest that anti-sRAGE autoantibodies may have a potential implication to evaluate metabolic risk and autoimmunity associated to RAGE

Recent statistics show that approximately 415 million people worldwide have diabetes. Glycated haemoglobin (HbA1c) measurements were introduced many years ago as the gold standard tool for detecting and monitoring treatment as well as making management decisions for diabetic patients. Glycated haemoglobins are formed by the non-enzymatic glycation of haemoglobin molecules. This non-enzymatic glycation process has been strongly related to pathogenesis of chronic complications associated to diabetes. It was suggested that this glycation process may be moderated by an enzymatic deglycation process thought to involve a deglycating enzyme known as Fructosamine-3-kinase (FN3K), an enzyme that deglycates the glycated haemoglobin in erythrocytes and other glycated proteins in other tissues. FN3K acts through phosphorylation of fructosamines on the third carbon of their sugar moiety, making them unstable and consequently causing them to detach from the protein. The degree of deglycation is thought to depend on the activity of the FN3K enzyme. Moreover, variation in the activity of FN3K between individuals is hypothesised to lead to apparent differences in glycated haemoglobin levels: some individuals have high rates of deglycation so that they tend to have lower average glycaemia than actually the case, while others with low rates of deglycation appear to have higher than actual glycaemia (known as the glycation gap, G-gap). The G-gap has been reported to be associated with alteration of diabetic complications risk. The G-gap reflects the discrepancy between average glycaemia as determined from glycated haemoglobin (measured as HbA1c) and that from the determination of fructosamine. The positive G-gap is defined as a higher level of glycation of proteins than expected whereas a negative G-gap means a lower level of glycation than expected. To explore the role of FN3K in diabetes and other associated morbidities, we decided to divide our research into 3 studies. Each study was categorised according to the type and the source of samples involved. The first study explored the correlation between FN3K activity and protein level with G-gap data; it involved 148 diabetic patients who were recruited at New Cross Hospital, Wolverhampton, selected as having a consistent positive G-gap > +0.5 and a consistent negative G-gap > -0.5 over a minimum of 2 estimations. Age, gender, race and BMI were collected from patients in this study. Blood samples were also 3 collected to measure FN3K activity, protein levels, and markers of CVD in relation to G-gap. The second study involved 23 AECOPD patients who were recruited from St George’s Hospital (London) and were treated with either metformin or a placebo. Serum samples were collected from these patients for a larger study: we assayed those 23 serum samples for FN3K protein levels to explore any possible correlation between FN3K with metformin therapy in COPD patients. The third study utilised 36 human peripheral lung samples from healthy individuals, asymptomatic smokers and stable COPD patients (GOLD 2) who were recruited at The Section of Respiratory Medicine, University Hospital of Ferrara, Italy. Those samples were assessed for FN3K expression by means of immunohistochemistry to explore the difference in FN3K activity between those three categories. It was found that the intracellular activity and protein expression of the FN3K enzyme in diabetic patients negatively correlated with the values of G-gaps where FN3K activity was high in patients with negative G-gap. FN3K serum protein levels were shown to be enhanced with metformin administration in COPD diabetic patients, suggesting a protective role for FN3K enzyme against protein damaged caused by the non-enzymatic glycation of proteins. Therefore, patients with positive G-gap have lower FN3K activity than those with negative G-gap, and in turn they are more susceptible to diabetes related complications. Our data also indicate that metformin has a beneficial effect in reducing damage caused by carbonyl stress from cigarette smoking in COPD patients by the action of FN3K. Our research has demonstrated that FN3K contributes to the protein repair system which protects against damage caused by non-enzymatic glycation. The high activity for the FN3K enzyme was associated with low levels of AGEs and low carbonyl stress levels in observed among patients with diabetes and COPD. In contrast, COPD patients tend to have low FN3K-mediated protection against protein damage in comparison to the normal population. These patients tend to be at risk for developing more complications, particularly CVD complications, than normal, healthy individuals. Treatment with metformin enhances FN3K action in COPD diabetic patients, possibly as a protective enzyme against the damaged caused by the non-enzymatic glycation.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-15T12:45:36Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Emanuela Paz Rosas Final.pdf: 1301979 bytes, checksum: 812637acf4c6404700fd08c4dbcc4009 (MD5) / Made available in DSpace on 2016-04-15T12:45:36Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Emanuela Paz Rosas Final.pdf: 1301979 bytes, checksum: 812637acf4c6404700fd08c4dbcc4009 (MD5) Previous issue date: 2015-08-27 / CAPEs / Os produtos finais de glicação avançada (AGEs) são compostos heterogêneos gerados a partir da reação não enzimática, irreversível, conhecida como reação de Maillard ou Glicação. Essa reação ocorre entre açúcares redutores e seus metabólitos reativos ou lipídeos oxidados e proteínas ou ácidos nucléicos. Os AGEs são formados endógena (hiperglicemia e estresse oxidativo) e exogenamente (alimentos e fumo), e podem ser acumulados em vários tecidos causando efeitos deletérios em diversas patologias relacionadas a idade e a diabetes. Uma de suas principais ações é gerar estresse oxidativo através do aumento da expressão de mediadores inflamatórios. Os macrófagos são células fundamentais no reconhecimento, degradação e remoção dessas substâncias, além disso, têm papel crucial na produção de espécies reativas de nitrogênio (RNS) na resposta imune, e podem estar envolvidos com variadas patologias. Neste estudo avaliou-se o efeito dos AGEs sobre a produção de óxido nítrico e a viabilidade celular em macrófagos. Macrófagos J774 foram incubados com diferentes concentrações de BSA- AGE (15, 30, 60, 120 ou 240μg/mL) por 24 ou 48 h e em seguida foram expostas (ou não) ao LPS (100ng/ml) por 24h. Para analisar a produção de óxido nítrico e a viabilidade celular utilizaram-se os métodos de Griess e de MTT, respectivamente. As células J774 submetidas ao tratamento com AGE apresentaram alterações nos níveis de NO e na viabilidade celular. Notou-se que os macrófagos quando incubados com AGE nas maiores concentrações (60, 120, 240μg/mL), sem LPS, produziram um aumento significativo na liberação de NO por 24 h. Também foi visto que o tratamento com AGE nas concentrações 120 e 240μg/mL, e com LPS, também elevaram os níveis de NO significativamente. Os resultados mostraram um aumento na produção de NO quando as células foram tratadas com AGE em todas as concentrações (15, 30, 60, 120 e 240μg/mL) por 48 h. A incubação de células J774 com AGE nas concentrações de 240 μg/ml por 24h com LPS, como também as concentrações de 120 e 240 μg/ml por 48h sem LPS, reduziram a viabilidade celular quando comparados com seus respectivos controles. Em conclusão, os AGEs podem causar aumento da produção de NO por macrófagos quando estes são estimulados ou não por LPS, e por consequência conduzir a morte celular. / The advanced glycation end products (AGEs) are heterogenic compounds generated from Maillard reaction or Glycation reaction. It is an irrevesible nonenzymatic reaction involving reductive sugars and reactive metabolites or oxidated lipids, proteins or nucleic acids. AGEs could be originated by endogenous way (hyperglycemia or oxidative stress) and by exogenous way (certain types of food and tobacco). They could be accumulated in several organic tissues causing deleterious effects seen in diabetes and age-related diseases. One of the main AGE effects is the induction and maintenance of stress oxidative through inflammatory mediators increasing expression. In this scenario, macrophages are fundamental cells on the recognition, degradation and clearance of AGEs. They also play an important role on immune response by stimulating reactive nitrogen species production (RNS), which could be involved on the evolution of several diseases. The present study evaluated the AGE effect on the macrophages nitric oxide production as well as the cell viability. J774 macrophages were incubated with different concentrations of BSA- AGE (15μg, 30μg, 60μg, 120μg ou 240μg/mL) by 24 or 48 hours, following a second incubation with or without LPS (100ng/mL) by 24 hours. Nitric oxide production was evaluated by Griess reaction and cell viability by MTT production. Macrophages incubated with AGE presented nitric oxide and cell viability alterations. It was noted that macrophages when incubated with AGE in higher concentrations (60, 120, 240μg/mL) without LPS produced a significant increase in the release of NO for 24 h. It has also been seen that treatment with AGE concentrations 120 and 240μg/mL and stimulation by LPS have also increased NO levels significantly. The results showed an increase in NO production when cells were treated with AGE at all concentrations (15, 30, 60, 120 and 240μg/ mL) for 48 h. The incubation J774 cells with AGE at concentrations of 240 μg/ mL for 24 hours with LPS, as well as the concentrations of 120 and 240 μg/ mL for 48h without LPS, reduced the cell viability compared to their respective controls. In conclusion, AGEs can cause increased production of NO by macrophages when these are stimulated or not with LPS, leading to cell death.

One of the greatest risk factors for disease is advanced age. As the human lifespan has increased, so too have the burdens of caring for an increasingly older population suffering from rising rates of cardiovascular disease, kidney disease, diabetes, and dementia. The need for improving medical technology and developing new therapies for age-related diseases is manifest. Yet our understanding of the processes of aging and how to attenuate the effects of aging remains incomplete. Various studies have established calorie restriction as a robust method for extending lifespan in laboratory organisms; however the mechanism is a topic of much debate. Advancing our understanding of calorie restriction holds promise for illuminating biochemical processes involved in the aging process. One of the best explanations for the lifespan extension benefits of calorie restriction is that it improves cellular protein homeostasis (proteostasis), but because proteostasis is dynamic, it can be difficult to measure. We developed a novel combined omics methodology integrating kinetic proteomics, and applied it to a mouse model placed on calorie restriction. Our unbiased approach integrating just three measurements (kinetic proteomics, quantitative proteomics, and transcriptomics) enabled us to characterize the synthesis and degradation of thousands of proteins, and determine that calorie restriction largely alters proteostasis by slowing global protein synthesis post-transcriptionally. Validating our omics approach, we were able to replicate many previous results found in the literature, demonstrating the differential regulation of various protein ontologies in response to the nutrient stress of calorie restriction. Moreover, we were able to detect differential degradation of the large and small ribosomal subunits under calorie restriction, and proposed a model in which the rate of protein synthesis could be attenuated by the depletion of the large ribosomal subunit relative to the small subunit. The flexibility of our dynamic combined omics approach was demonstrated by the expansion of measurements to include nucleic acids and lipids. Flux measurements of DNA, ribosomal RNA, and lipids yielded cellular division rates, ribosome turnover, and lipid metabolism insights, respectively. We also adapted this approach to two-dimensional tissue imaging by DESI-MS in a proof-of-concept study to demonstrate its utility for studying regional differences in metabolism. The future integration of metabolomics and lipidomics into our combined omics approach would be facile, and add unprecedented depth to systems-wide studies involving cellular metabolism. Applied to the regulation of cellular homeostasis in humans, this has the potential to open new avenues for elucidating the etiology of aging, understanding the pathology of age-related diseases, and identifying novel targets for therapeutics.