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.

Cyclooxygenase (COX) is the rate limiting enzyme that catalyses the production of prostanoids, which are crucial to vascular homeostasis. Evidence suggests that endothelial dysfunction and inflammation play a role in vascular complications in aging and diabetes. Previous animal studies by our laboratory at RMIT University reported enhanced COX expression with aging in rat aortas, platelets and monocytes. Potentially, alteration in COX expression may result in an imbalanced prostanoid production favoring the synthesis of vasoconstrictors and hence increase the risk of cardiovascular events in the aging population. The regulation of altered COX expression in aging, however, is not clear. It has been suggested that histone hyperacetylation may be an important mechanism that regulates COX levels during the aging process as increased histone acetylation has been shown to occur with aging. Thus, we hypothesized that COX expression is modulated by histone hyperacetylati on. This was investigated by measuring COX expression in histone hyperacetylated cultured endothelial cells. In the case of diabetes, studies have reported that the development of diabetes and its complications is associated with persistent inflammatory activity, evident with increased inflammatory markers in the circulation. COX-mediated pathways may be involved in this inflammatory process in diabetes. Furthermore, the formation of advanced glycation end products (AGEs) is accelerated in diabetes. AGEs can bind to receptors for AGEs (RAGE), which has also been suggested to play a role in inflammation in diabetes. We hypothesized that COX- and RAGE-mediated pathways contribute to increased inflammation in diabetes and potentiate the development of diabetic vascular complications. This was investigated by measuring changes in COX-mediated pathways in both rat and human diabetic models. The current thesis reports: 1) in cultured endothelial cells, histone hyperacetylation was associated with increased COX expression; 2) an overall increase in inflammation was observed in diabetes involving COX- and RAGE-mediated pathways. This was supported by increased platelet COX-1 and monocyte COX-2 levels in Zucker rats, increased monocyte COX-2 in human Type 1 diabetes and elevated plasma TXB2 and PGE2 levels in both human Type 1 and Type 2 diabetic subjects. Up-regulation of RAGE expression was further found in platelets and monocytes in both human diabetes types. When treated with NSAIDs, plasma prostanoid levels, COX and RAGE expression were reduced significantly in both platelets and monocytes in human diabetic subjects. 3) It is unclear how COX and RAGE expression was regulated, but histone modifications may be one of the mechanisms. Data from cultured cells indicated that increased COX expression was associated with increased histone acetylation levels induced by TSA. Concurrent increases in histone acetylation and COX-2 levels were also observed in human Type 1 diabetes, but similar findings were not observed in human Type 2 diabetes. In addition, we failed to find an age-dependent increase in monocyte histone H4 acetylation in human Type 2 diabetes despite an age-dependent increase in monocyte COX-2 expression. Thus, whether histone hyperacetylation modulates COX expression and in what conditions require further investigation.

Dans un contexte de vieillissement de la population et d’accroissement des maladies chroniques liées à l’âge comme le diabète, de nouveaux biomarqueurs de l’état de santé à long terme doivent être étudiés. Les produits de glycation avancée (AGE) sont des molécules témoins de la charge métabolique accumulée au cours du temps, dénommée "mémoire métabolique". Les AGE jouent un rôle important dans les lésions à long terme dans le diabète et dans le déclin du métabolisme global lié au vieillissement. L’accumulation cutanée des AGE peut être mesurée par autofluorescence (AF) de manière instantanée et non invasive grâce à l’AGE-READER. Les objectifs de cette thèse étaient d’évaluer la valeur de l’AF cutanée des AGE en tant que marqueur de mémoire métabolique chez des personnes âgées de la cohorte des 3-Cités et parallèlement d’évaluer la valeur pronostique de l’AF pour les complications du diabète chez des patients porteurs de diabète de type 1. Chez les personnes âgées, nous avons montré que l’AF reflétait les statuts glycémique et rénal 10 ans avant la mesure. Chez les patients atteints de diabète de type 1, l’AF était associée à la présence d’une neuropathie 4 ans plus tard. De plus, dans cette même population, nous avons décrit l’évolution de l’AF sur 4 ans de suivi. Nous avons montré que les principaux déterminants de son évolution étaient la fonction rénale et le traitement par pompe à insuline. Enfin nous avons trouvé que l’augmentation de l’AF sur 4 ans de suivi était associée à la survenue de la maladie rénale. Ces travaux soulèvent de nouvelles perspectives de recherche quant à l’intérêt de l’AF à différents âges clés de la vie en tant que biomarqueur de pathologies qui évoluent sur des dizaines d’années. / In the context of the ageing of the population and the increase of age related diseases such as diabetes, new biomarquers of the long-term health status should be considered. Advanced glycation end products (AGE) are molecules indicators of the metabolic burden over time, called “metabolic memory”. AGE play an important role in long term diabetes injuries and in the global decline of the metabolism related to ageing. Skin accumulation of AGE can be measured by autofluorescence instantly and non-invasivly with a tool called AGE-READER. The objectives of my dissertation were to evaluate the value of the skin autofluorescence (sAF) of AGE as marker of metabolic memory in elderly people from the 3-City cohort and in parallel, in patients with type 1 diabetes, evaluate the prognostic value of sAF for diabetes complications. In the elderly population, we showed that sAF reflected glycemic and renal status of 10 years before. In patients with type 1 diabetes, sAF was associated to the presence of neuropathy 4 years later. Moreover, in this same population, we described the evolution of sAF in 4 years of follow-up and we showed that the principal determinants of the evolution of sAF were kidney function and insulin pump therapy. Finally, we also found that increase of sAF in 4 years was associated with the occurrence of kidney disease. This work rises new research opportunities about the interest of sAF at differents key ages as biomarker of pathologies which evolve in several decades.