Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Introduction: The discovery of the endothelium as a regulator of vascular tone, and the subsequent discovery of nitric oxide (NO) as the major endothelium-derived relaxing factor (EDRF), has opened up vast possibilities in the continued efforts to prevent and manage cardiovascular disease. Endothelial dysfunction (ED) is defined as reduced NO bioavailability and hence the reduced ability of the endothelium to maintain vascular homeostasis. ED represents the first, reversible step in the initiation of atherosclerotic disease and is thus regarded as a strong predictive tool of ischaemic heart disease (IHD). ED and its underlying mechanisms have been largely under-investigated in myocardial capillary-derived endothelial cells (cardiac microvascular endothelial cells, CMECs), and this study aimed to address this gap in the literature. Oleanolic acid (OA) is a bioactive triterpenoid derived from leaf extracts of African medicinal plants such as Syzigium cordatum (Water berry tree), and has been reported to elicit vasodilatory, hypoglycaemic and hypolipidaemic properties. However its effects particularly on CMECs and its putative role in reversing ED remain unclear, and this study aimed to investigate such effects. Aims: The aims of this study were to: (1) Establish an in vitro model of ED in cultured myocardial capillary-derived CMECs by developing protocols for the induction of ED. (2) Asses ED induction by measurement of the following biomarkers: (i) intracellular NO production, (ii) superoxide (O2-) production, (iii) nitrotyrosine expression and (iv) NADPH oxidase expression. (3) Investigate underlying cellular mechanisms of our ED model by measuring and comparing eNOS and PKB/Akt expression and activation in control and dysfunctional CMECs. (4) Investigate the effects of OA derived from leaf extracts obtained from Syzigium cordatum (Hochst.) [Myrtaceace], in both control and dysfunctional CMECs. Methods: (1) To induce ED, hyperglycaemia and inflammation were simulated by incubation with 25 mM glucose (24 hours) and 1 ng/ml TNF-á (24 hours) or 5 ng/ml TNF-á (6 and 24 hours) respectively. Reduced intracellular NO production was used as the main indicator of ED. NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide (PI) / Annexin V respectively. Cellular mechanisms were investigated by measurement of O2- levels via FACS analysis of DHE fluorescence, and measurement of total and activated PKB / Akt and eNOS, p22-phox, nitrotyrosine expression via Western blotting. (2) Effects of OA on CMECs were investigated by pre-treatment with 30 or 40 ìM OA for 5 and 20 min followed by NO production and cell viability measurements. To investigate the effects of OA on ED, CMECs were pre-treated with 40 ìM OA 1 hour prior ED induction followed by NO, cell viability, and eNOS expression / activation measurements. Results: (1) 25 mM glucose (24hours), 1 ng/ml TNF-á (24 hours) and 5 ng/ml TNF-á (6 hours) failed to induce ED as verified by an increase in NO production in the treated cells. A model of ED was successfully achieved by incubating CMECs with 5 ng/ml TNF-á (24 hours), as verified by a significant decrease in NO production. Investigations into cellular mechanisms underlying our TNF-á-induced ED model, showed that activated eNOS and PKB / Akt levels were reduced. Furthermore, O2- levels remained unchanged, however p22-phox (NADPH) expression was significantly increased suggesting oxidative stress. Nitrotyrosine levels (an oxidative / nitrosative stress marker and indirect measure of eNOS uncoupling) remained at control levels. (2) Investigations into the effects of OA on CMECs showed that 30 ìM OA increased NO production after 5 and 20 min of incubation whereas 40 ìM increased NO production after 20 min only. Pre-treatment with 40 ìM OA significantly reversed ED by restoring NO production back to control levels. Data from cellular mechanism investigations showed that 40 ìM OA significantly increased eNOS activation in both normal and dysfunctional CMECs. Cellular viability was not negatively affected by any of the above interventions. Discussion and Conclusions: Based on our findings, reduced activation of the PKB / Akt-eNOS pathway appears to be the primary mechanistic pathway of the TNF-á-induced model of ED. Though O2- levels remained at control levels, the significant increase in p22-phox is indicative of increased expression of the O2- producing enzyme, NADPH oxidase, thus suggesting oxidative stress. However, based on our nitrotyrosine expression data, there was no strong evidence of eNOS uncoupling in our ED model. OA significantly stimulated NO production in our model of CMECs. Furthermore, our findings showed that OA is able to reverse ED. The NO production stimulatory effects of OA in our cells appear to be achieved via the increased activation of eNOS. We have, for the first time as far as we are aware, developed a TNF-á-induced model of ED in myocardial capillary-derived endothelial cells. It appears that reduced activation of the PKB/Akt-eNOS pathway is the primary mechanism leading to decreased NO production in this model. However, we did find some evidence of elevated oxidative stress, which led us to believe that eNOS uncoupling cannot be excluded as a mechanism of ED in our model. In this study, we report for the first time convincing evidence that OA has powerful NO-increasing properties in myocardial capillary-derived CMECs. Our study also show novel data, which suggest that OA is able to reverse ED in this model. Follow-up investigations could shed more light on the exact mechanisms underlying OA.s effects in this model. / AFRIKAANSE OPSOMMING: Inleiding: Die ontdekking dat endoteel 'n reguleerder van vaskulêre tonus is, en die gevolglike ontdekking dat stikstofoksied (NO) die belangrikste endoteel-afgeleide verslappingsfaktor (EDRF) is, het verskeie moontlikhede in aangaande pogings om kardiovaskulêre siektes te voorkom en hanteer, ontsluit. Endoteel-disfunksie (ED), word gedefineer as verlaagde NO biobeskikbaarheid en dus 'n ingekorte vermoë van die endoteel om vaskulêre homeostase te handhaaf. ED verteenwoordig die eerste, omkeerbare stap in die ontstaan van aterosklerotiese siekte en word dus beskou as 'n sterk instrument waarmee isgemiese hartsiekte voorspel kan word. Studies oor ED en sy onderliggende meganismes, veral in miokardiale kapillêre-afgeleide endoteelselle (kardiale mikrovaskulêre endoteelselle, CMECs), word redelik afgeskeep in die literatuur, en hierdie studie het dit ten doel gehad om die gaping in die literatuur aan te spreek. Oleanoliese suur (OA) is 'n bio-aktiewe triterpenoïede wat gevind word in blaar ekstrakte van inheemse medisinale plante soos bv. Syzigium cordatum (Waterbessie boom). OA het bewese vasodilatoriese, hipoglukemiese en hipolipidemiese eienskappe. OA se effekte op CMECs, en sy moontlike rol in die omkering van ED, is egter onbekend, en hierdie studie het dit ten doel gehad om sulke effekte te ondersoek. Doelwitte: Die doelwitte van hierdie studie was: (1) Die vestiging van 'n in vitro model van ED in gekultuurde CMECs afkomstig van miokardiale kapillêre deur protokolle vir die induksie van ED te ontwikkel. (2) Die evaluering van ED induksie deur die volgende bio-merkers te meet: (i) intrasellulêre NO produksie, (ii) superoksied (O2-) produksie, (iii) nitrotirosien uitdrukking en (iv) NADPH oksidase uitdrukking. (3) Die ondersoek na onderliggende sellulere meganismes van ED in ons model deur die meting en vergelyking van eNOS and PKB/Akt uitdrukking en aktivering in kontrole en disfunksionele CMECs. (4) Ondersoek na die effekte van OA afkomstig van blaar ekstrakte verkry van Syzigium cordatum (Hochst.) [Myrtaceace], in beide kontrole en disfunksionele CMECs. Metodes: (1) Daar was gepoog om ED te induseer deur hiperglukemie en inflammasie te simuleer met onderskeidelik 25 mM glukose (24 uur) en 1 ng/ml TNF-a (24 uur) of 5 ng/ml (6 en 24 uur) inkubasie. Verlaagde intrasellulere NO produksie was ingespan as die hoof indikator van ED. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied (PI) / Annexin V onderskeidelik. Sellulere meganismes was ondersoek deur O2- vlakke via FACS analise van DHE fluoressensie te meet, asook die meting van totale en geaktiveerde PKB / Akt en eNOS, p22-phox, nitrotirosien uitdrukking via Western blot tegnieke. (2) Effekte van OA op CMECs was ondersoek deur vooraf-behandeling met 30 of 40 µM OA vir 5 en 20 min gevolg deur NO produksie en sellewensvatbaarheid metings. Resultate: (1) 25 mM glukose (24 uur), 1 ng/ml TNF-a (24 uur) and 5 ng/ml TNF-ƒaa (6 uur) kon nie daarin slaag om ED te induseer nie, soos blyk uit die verhoogde NO produksie waargeneem in die behandelde selle. 'n Model van ED was suksesvol verkry deur CMECs met 5 ng/ml TNF-a (24 uur) te inkubeer, soos waargeneem deur verlaagde NO produksie. Ondersoek na sellulere meganismes onderliggend tot ons TNF-a-geinduseerde ED model, het getoon dat geaktiveerde eNOS en PKB / Akt vlakke verlaag was. Verder is gevind dat O2- vlakke onveranderd gebly het hoewel p22-phox (NADPH) uitdrukking betekenisvol toegeneem het, wat 'n aanduiding van oksidatiewe skade is. Nitrotirosien vlakke (.n oksidatiewe / nitrosatiewe stres merker en indirekte maatstaf van eNOS ontkoppeling) het onveranderd rondom kontrole vlakke gebly. (2) Ondersoek na die effekte van OA op CMECs het getoon dat 30 µM OA tot verhoogde NO produksie na 5 en 20 min inkubasie gelei het, terwyl 40 µM slegs na 20 min NO-verhogende effekte gehad het. Vooraf behandeling met 40 µM OA het ED betekenisvol omgekeer deur NO terug na kontrole vlakke te laat herstel. Ondersoek na sellulere meganismes het getoon dat 40 µM OA eNOS aktivering betekenisvol verhoog het in beide normale en disfunksionele CMECs. Sellulere lewensvatbaarheid was nie negatief geaffekteer deur enige van bogeneemde ingrepe nie. Bespreking en afleidings: Gebaseer op ons bevindinge, blyk verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganistiese pad in ons TNF-a-geïnduseerde model van ED te wees. Alhoewel O2- vlakke rondom kontrole vlakke gebly het, was die betekenisvolle toename in p22-phox .n aanduiding van verhoogde uitdrukking van die O2- produserende ensiem, NADPH oksidase, wat dus suggererend van oksidatiewe stres was. Aan die ander kant was daar nie sterk bewyse van eNOS ontkoppeling in ons ED model nie, gebaseer op die nitrotirosien uitdrukking data. OA het duidelik NO produksie in ons model van CMECs gestimuleer. Verder wys ons resultate dat OA in staat is om ED om te keer. Die NO produksie-stimulerende effekte van OA in ons selle blyk die gevolg te wees van verhoogde aktivering van die PKB / Akt-eNOS pad. Ons het hier vir die eerste keer, sover ons bewus is, 'n TNF-a-geinduseerde model van ED in CMECs afkomstig van miokardiale kapillere gevestig. Dit blyk dat verlaagde aktivering van die PKB/Akt-eNOS pad die primere meganisme was waardeur verlaagde NO produksie in ons model veroorsaak was. Ons het egter wel bewyse van verhoogde oksidatiewe stress gevind, wat ons laat glo dat eNOS ontkoppeling nie heeltemal as .n meganisme van ED in ons model uitgesluit kan word nie. In hierdie studie toon ons vir die eerste maal oortuigende bewyse dat OA kragtige NO-verhogende eienskappe in miokardiale kapillere-afgeleide CMECs het. Ons studie bring ook nuwe data na vore, wat suggereer dat OA in staat is om ED in hierdie model om te keer. Opvolgstudies sal meer lig kan werp op die onderliggende meganismes van OA in hierdie model.

L'hyperhomoscystéinémie (HHcy) est considérée comme un facteur indépendant de risque cardio-métabolique. Notre travail avait pour objectifs : 1) de déterminer la prévalence de l’HHcy au Bénin; 2) d’étudier sa relation avec les apports de vitamines B12, B9, B6 et B2, la consommation d’alcool, l’âge, le sexe et le niveau socioéconomique (NSE); 3) de vérifier son association avec les facteurs classiques de risque cardio-métabolique. Un total de 541 sujets apparemment en santé et vivant dans trois zones du Bénin ont été étudiés. L’Hcy sérique a été analysée par ELISA. Des rappels de 24h ont servi à évaluer les apports nutritifs. L’obésité et l’hypertension ont été définies selon l’OMS, la dyslipidémie et la dysglycémie selon la NCEP-ATPIII. Les autres données ont été récoltées par questionnaire. La prévalence de l’HHcy était élevée : 52,2% chez les hommes et de 24,7% chez les femmes. Dans les modèles multivariés, l’Hcy était positivement associée à la consommation de bière locale chez les hommes; chez les femmes, elle était associée négativement à l'apport de vitamine B12. L’Hcy était positivement associée à la tension artérielle, au taux de LDL-cholestérol et au cholestérol total chez les hommes, mais seulement avec le rapport cholestérol total/HDL-cholestérol (CT/HDL-c) chez les femmes. Les femmes présentant une HHcy étaient au moins deux fois plus susceptibles de présenter une hypertension ou un rapport CT/HDL-c élevé que celles dont l’Hcy était normale. Un apport suffisant en B12 ainsi qu’une consommation prudente de boissons alcoolisées pourraient réduire l'HHcy et donc contribuer à réduire le risque cardio-métabolique de cette population du sud du Bénin. / Hyperhomocysteinemia (HHcy) appears to be an independent risk factor for cardiovascular disease. This study aims to determine the prevalence of HHcy in Benin, to explore its relationship with intakes of folate, B12, B6 and B2, with alcohol consumption and with socioeconomic status (SES) and to verify whether it is associated with classical risk factors of cardiovascular diseases. 541 apparently healthy subjects were randomly selected in tree areas of Benin. Hcy was measured in serum using ELISA commercial kits. Nutrient intakes were assessed on the basis of three non-consecutive 24-hour recalls. Alcohol consumption, socio-demographics and SES were documented in personal interviews. Obesity, hypertension, dyslipidemia and hyperglycaemia were defined according to WHO and NCEP-ATPIII. HHcy (> 12 μmol/L) was detected in 52.2% of men and 24.7% of women. In multivariate models, Hcy in men was positively associated with alcohol intake, but only alcohol in beer. In women, Hcy was negatively related to vitamin B12 intake. HHcy was associated in women with more than twice the odds of hypertension and with the CT/HDL-c ratio. In men, Hcy was positively and independently associated with diastolic blood pressure and with LDL-cholesterol and total cholesterol. In this Beninese population, the prevalence of HHcy is particularly high among men, and it appears to be related to alcohol consumption. Inadequate intake of vitamin B12 may be a risk factor for HHcy which could be related to some of cardiovascular factors.

L'insuffisance rénale chronique (IRC) demeure un problème de santé publique du fait de l'augmentation de sa prévalence. Malgré l'amélioration de la prise en charge, le taux de mortalité reste plus élevé comparé à la population générale. Parmi les causes de décès, les maladies cardiovasculaires, d'origine multifactorielle (élargissement et hypertrophie des artères, athérosclérose, calcifications vasculaires et valvulaires) sont au premier plan. A côté des facteurs de risque classiques, des facteurs non traditionnels, liés aux perturbations métaboliques de l'IRC, ont été mis en évidence, comme l'inflammation, la malnutrition, le stress oxydant, les anomalies du métabolisme minéralo-osseux. La meilleure connaissance de la physiopathologie de la vasculopathie de l'IRC permet d'émerger de nouveaux biomarqueurs pour stratifier le risque cardiovasculaire chez l'IRC.OBJECTIFS-METHODOLOGIE GENERALE : Nous avons réalisé une approche biochimique pour explorer trois composantes du risque cardiovasculaire chez l'IRC : stress oxydant, perturbations qualitatives des HDL (high-density lipoprotein) et métabolisme minéralo-osseux.RESULTATS : Dans une première publication la production d'anion superoxyde a été évaluée, via une méthode de chemoluminescence, en fonction du stade de l'IRC. Alors que la surproduction de formes réactives de l'oxygène est bien connue au stade 5d et peut être liée à la procédure dialytique, il existe peu de données aux stades précoces. Notre étude a porté sur 136 patients IRC non dialysés des stades 1à 5. Les résultats montrent que la production de FRO est assurée aux stades 4 et 5. Un bas débit de filtration glomérulaire (MDRD<30ml/min/1.73m2), l'inflammation (fibrinogène >3.7g/l) et des taux anormaux d' HDL (<1.42mM et >1.75mM) apparaissent comme les principaux déterminants du stress oxydant chez l'IRC non dialysé.Alors que dans la population générale, un taux bas de HDL est reconnu comme un facteur de risque important, nous avons montré (publication 1) que des taux anormaux de HDL, bas comme hauts, étaient indépendamment associés au stress oxydant chez les sujets IRC. Dans une deuxième publication, nous avons précisé la composition des HDL en se basant sur d'éventuelles modifications qualitatives des protéines associées à la structure des lipoprotéines. Une étude protéomique a été réalisée chez 7 patients hémodialysés versus 7 sujets sains. Nous avons retrouvé 40 protéines exprimées différemment sur les 122 identifiées, dont l'apoCII, l'apoCIII qui sont significativement augmentées et la transferrine abaissée. Ces protéines interviennent dans de nombreuses fonctions comme la réponse inflammatoire, l'activation du complément, la régulation de l'oxydation des lipoprotéines, l'homéostasie des cations.Dans une troisième publication, l'épuration du FGF23, phosphatonine impliquée dans les anomalies du métabolisme minéralo-osseux, été étudiée chez l'hémodialysé chronique en fonction de la techniques de dialyse (hémodialyse (HD) high flux versus hémodiafiltration on line (OL-HDF)). Notre étude a porté sur 53 patients dans le groupe HD et 32 patients dans le groupe OL-HDF. Dans les deux groupes le taux de FGF23 en post-dialyse est significativement plus bas qu'en pré-dialyse. Cependant, le taux de réduction, la clairance et le KT/V du FGF23 sont significativement plus bas dans le groupe OL-HDF.CONCLUSION-PERSPECTIVES : Chez l'IRC, avec l'appariation de facteurs de risque non traditionnels, de nouveaux biomarqueurs ont émergés dans la stratification du risque cardio-vasculaire. Ces biomarqueurs peuvent devenir des bioacteurs et représenter de nouvelles cibles d'action et de prévention de l'atteinte cardio-vasculaire chez l'IRC. La complexité des mécanismes physiopathologiques impliqués, nous incite à proposer des approches multimarqueurs. Actuellement des études biocliniques se poursuivent en mettant en place des cohortes régionales de patients aux stades 1 à 5 et de patients incidents en dialyse. / BACKGROUND: Chronic kidney disease (CKD) is a public health problem because of its increasing prevalence. Despite care improvements, the mortality rate remains higher compared to general population. Among causes of death, cardiovascular diseases with multifactorial origins (enlargement and hypertrophy of arteries, atherosclerosis, vascular and valvular calcifications) are in the foreground. Besides the traditional risk factors, non-traditional factors associated with metabolic disorders in CKD were bring out, such as inflammation, malnutrition, oxidative stress, mineral and bone disorder. A better knowledge of vasculopathy physiopathology in CKD allows the emergence of new biomarkers to stratify cardiovascular risk in CKD.AIMS-METHODOLOGY: We performed a biochemical approach to explore three components of cardiovascular risk in CKD: oxidative stress, qualitative alterations of HDL (high-density lipoprotein) and mineral and bone disorder.RESULTS: In a first publication, the superoxide anion production, according to the stage of CKD, was assessed using a chemiluminescence method. While the overproduction of reactive oxygen species is well known at the 5d stage of CKD and may be related to the dialysis procedure, there are few data in the early stages. Our study included 136 non-dialysis patients at stages 1 to 5 of CKD. Results showed an enhanced superoxide production at the pre-dialysis phase, stages 4 and 5 of CKD. Reduced glomerular filtration rate (MDRD <30 ml / min / 1.73m2), inflammation (fibrinogène≥3.7g / l) and abnormal levels of HDL (<1.42mM and ≥1.75mM) appears as main determinants of oxidative stress in non-dialysis CKD patients.While in general population, a low HDL rate is recognized as an important risk factor, we showed (publication 1) that abnormal levels of HDL, low as high, were independently associated with oxidative stress in CKD subjects. In a second publication, we have defined the HDL composition based on qualitative changes in the structure of proteins associated with lipoproteins. A proteomic study was performed in 7 patients on hemodialysis versus 7 healthy subjects. We found 40 proteins differently expressed on the 122 identified, including apoCII, apoCIII which are significantly increased and transferrin lowered. These proteins are involved in many functions such as inflammatory response, complement activation, regulation of lipoprotein oxidation and homeostasis cations. In a third publication, the removal of FGF23, phosphatonin involved in mineral and bone metabolism, was studied in chronic hemodialysis according to the dialysis techniques (high flux hemodialysis (HD) versus on line hemodiafiltration (OL- HDF)). Our study included 53 patients in the HD group and 32 patients in the OL-HDF group. In both groups the rate of FGF23 in post-dialysis was significantly lower than in pre-dialysis. However, rate of reduction, clearance and KT / V of FGF23 were significantly lower in the OL-HDF group.CONCLUSION-PROSPECTS: In the IRC, with the appearance of non traditional risk-factors, new biomarkers have emerged in the stratification of cardiovascular risk. These biomarkers can become bioactors and represent novel targets of action and prevention in the cardiovascular disease in CKD. The complexity of the involved physiopatholological mechanisms, leads us to propose multimarkers approaches. Currently bioclinical studies continue with the constitution of regional cohorts of patients at stages 1 to 5 of CKD and incident dialysis.

Combination therapy has proven to be a popular treatment strategy for tighter diabetes control. Since the preliminary evidence is suggestive of complementary actions of American (AG) and Korean Red Ginseng (KRG) in improving glycemia, this project was designed to investigate the therapeutic potential of AG and KRG in combination. Following a randomized, double-blind, placebo-controlled parallel design in a population with diabetes at two centres, the combined use of AG and KRG for 12 weeks was safe, but did not significantly affect glycemic control, blood lipids or blood pressure. However, there was a trend toward lower glycated hemoglobin by 0.7% (p=0.1) and office systolic blood pressure by 5 mm Hg (p=0.052) compared to placebo. These findings encourage further investigation of the mechanism and roles of AG, KRG and their effective components. They also highlight limitations in ginseng research and the need to impose strict regulations to facilitate its standardization.

Combination therapy has proven to be a popular treatment strategy for tighter diabetes control. Since the preliminary evidence is suggestive of complementary actions of American (AG) and Korean Red Ginseng (KRG) in improving glycemia, this project was designed to investigate the therapeutic potential of AG and KRG in combination. Following a randomized, double-blind, placebo-controlled parallel design in a population with diabetes at two centres, the combined use of AG and KRG for 12 weeks was safe, but did not significantly affect glycemic control, blood lipids or blood pressure. However, there was a trend toward lower glycated hemoglobin by 0.7% (p=0.1) and office systolic blood pressure by 5 mm Hg (p=0.052) compared to placebo. These findings encourage further investigation of the mechanism and roles of AG, KRG and their effective components. They also highlight limitations in ginseng research and the need to impose strict regulations to facilitate its standardization.

In den ersten Versuchsreihen der hier vorliegenden Arbeit bestätigte sich, dass im in vitro Modell die klassische ischämische Präkonditionierung kardioprotektiv wirkt. Die Präkonditionierung bewirkte eine Infarktgrößenreduktion um 54 %; dies wird durch Literaturangaben bestätigt. Die Postkonditionierung dagegen, trotz drei verschiedener Postkonditionierungsprotokolle, ist am isoliert perfundierten Rattenherzen nicht protektiv. Im in vivo Rattenherz-Modell wurden die Präkonditionierung und die klinisch relevantere Postkonditionierung gegenüberstellend untersucht. Hier zeigte sich, dass die 3 Reperfusions-/Ischämiezyklen für jeweils 30 Sekunden der Postkonditionierung genauso protektiv wie die Präkonditionierung wirken. Infarktgrößen- und biochemische Untersuchungen belegen, dass hierbei die PI3-Kinase ein wichtiges Signaltransduktionselement ist, da einerseits durch die Inhibition der PI3-Kinase mittels Wortmannin die Infarktgrößenreduktion vollständig aufgehoben war und andererseits nach einer 1,5-minütigen Reperfusion eine vermehrte Phosphorylierung der Akt im Western-Blot auftrat. Des Weiteren konnte erstmals die Inaktivierung der GSK-3ß durch eine verstärkte Phosphorylierung über einen PI3-Kinase-vermittelten Signaltransduktionsweg nachgewiesen werden. Die Zugabe des spezifischen Inhibitors TDZD-8 der GSK-3ß verringert ebenfalls die Infarktgröße signifikant. Auch konnte zum ersten Mal gezeigt werden, dass das mammalian target of Rapamycin in der Postkonditionierung des in vivo Rattenherzens eine wichtige Rolle zu spielen scheint. Außerdem konnte neben dem PI3-Kinase/Akt-Signaltransduktionsweg auch die Beteiligung des MEK1/2-ERK1/2–Wegs als Signaltransduktionsweg der Postkonditionierung im in vivo Rattenherzen nachgewiesen werden. Erstmals wurde die Apoptose in einem in vivo Herzen nach regionaler Ischämie untersucht. Die Ergebnisse des TUNEL-Tests und der Western-Blot-Analysen zeigen eine unterdrückte Apoptose durch die Postkonditionierung. Ein weiterer Teil der vorliegenden Arbeit widmete sich der Untersuchung der Postkonditionierung in pathologischen Rattenherzen. Im Gegensatz zu gesunden Herzen schlug die Postkonditionierung in hypertrophiertem Myokardium von spontan-hypertensiven Ratten mit einer signifikant arteriellen Hypertension fehl. Diese Blockierung der Kardioprotektion zeigte sich durch die fehlende Reduzierung der Infarktgröße trotz unterschiedlicher Postkonditionierungsprotokolle (3x30’’ und 6x10’’ R/I) und unterschiedlich langer Ischämiedauern (20 und 30 Minuten). Gleichfalls war auch die Phosphorylierung der GSK-3ß aufgehoben. Als Modell des metabolischen Syndroms wurde die WOKW-Ratte untersucht. Diese Ratten entwickeln in sehr jungem Alter klassische Symptome wie Dyslipidämie, Hyperinsulinämie und Fettsucht. Wie bei der Herzhypertrophie war auch beim Modell des metabolischen Syndroms die Postkonditionierung - mit 3 Reperfusions-/ Ischämiezyklen für jeweils 30 Sekunden - blockiert. Dabei konnte weder eine Infarktgrößenreduktion noch eine vermehrte Phosphorylierung der GSK-3ß nachgewiesen werden. Die Ergebnisse der vorliegenden Arbeit erlauben die Schlußfolgerung, dass das Substrat der GSK-3ß, die mPTP des Mitochondriums, eine „Schlüsselrolle“ in der Apoptose innehat - die Postkonditionierung vermindert nicht nur die Nekrose, sondern reduziert auch die Apoptose. Bemerkenswert und potentiell von klinischer Bedeutung ist die Beobachtung, dass bei Vorliegen von Risikofaktoren, wie arterielle Hypertonie und metabolischem Syndrom, solche Schutzmechanismen des Herzens aufgehoben sind. Diese Erkenntnisse sind im Hinblick auf die Therapie am Menschen von großer Bedeutung. Ob langfristig einzelne Komponenten der Signaltransduktionswege, wie PI3-Kinase, Akt, mTOR, ERK1/2 oder GSK-3ß, Angriffspunkte einer pharmakologischen Therapie sein könnten, muß in weiteren Untersuchungen geklärt werden.

L'hyperhomoscystéinémie (HHcy) est considérée comme un facteur indépendant de risque cardio-métabolique. Notre travail avait pour objectifs : 1) de déterminer la prévalence de l’HHcy au Bénin; 2) d’étudier sa relation avec les apports de vitamines B12, B9, B6 et B2, la consommation d’alcool, l’âge, le sexe et le niveau socioéconomique (NSE); 3) de vérifier son association avec les facteurs classiques de risque cardio-métabolique. Un total de 541 sujets apparemment en santé et vivant dans trois zones du Bénin ont été étudiés. L’Hcy sérique a été analysée par ELISA. Des rappels de 24h ont servi à évaluer les apports nutritifs. L’obésité et l’hypertension ont été définies selon l’OMS, la dyslipidémie et la dysglycémie selon la NCEP-ATPIII. Les autres données ont été récoltées par questionnaire. La prévalence de l’HHcy était élevée : 52,2% chez les hommes et de 24,7% chez les femmes. Dans les modèles multivariés, l’Hcy était positivement associée à la consommation de bière locale chez les hommes; chez les femmes, elle était associée négativement à l'apport de vitamine B12. L’Hcy était positivement associée à la tension artérielle, au taux de LDL-cholestérol et au cholestérol total chez les hommes, mais seulement avec le rapport cholestérol total/HDL-cholestérol (CT/HDL-c) chez les femmes. Les femmes présentant une HHcy étaient au moins deux fois plus susceptibles de présenter une hypertension ou un rapport CT/HDL-c élevé que celles dont l’Hcy était normale. Un apport suffisant en B12 ainsi qu’une consommation prudente de boissons alcoolisées pourraient réduire l'HHcy et donc contribuer à réduire le risque cardio-métabolique de cette population du sud du Bénin. / Hyperhomocysteinemia (HHcy) appears to be an independent risk factor for cardiovascular disease. This study aims to determine the prevalence of HHcy in Benin, to explore its relationship with intakes of folate, B12, B6 and B2, with alcohol consumption and with socioeconomic status (SES) and to verify whether it is associated with classical risk factors of cardiovascular diseases. 541 apparently healthy subjects were randomly selected in tree areas of Benin. Hcy was measured in serum using ELISA commercial kits. Nutrient intakes were assessed on the basis of three non-consecutive 24-hour recalls. Alcohol consumption, socio-demographics and SES were documented in personal interviews. Obesity, hypertension, dyslipidemia and hyperglycaemia were defined according to WHO and NCEP-ATPIII. HHcy (> 12 μmol/L) was detected in 52.2% of men and 24.7% of women. In multivariate models, Hcy in men was positively associated with alcohol intake, but only alcohol in beer. In women, Hcy was negatively related to vitamin B12 intake. HHcy was associated in women with more than twice the odds of hypertension and with the CT/HDL-c ratio. In men, Hcy was positively and independently associated with diastolic blood pressure and with LDL-cholesterol and total cholesterol. In this Beninese population, the prevalence of HHcy is particularly high among men, and it appears to be related to alcohol consumption. Inadequate intake of vitamin B12 may be a risk factor for HHcy which could be related to some of cardiovascular factors.

Atherosclerotic cardiovascular diseases (CVD), particularly coronary heart disease (CHD) and cerebrovascular disease, are today major causes of death in the industrialised parts of the world. There are evidence to suggest that the atherosclerotic process starts in childhood, implying that preventive measures should be implemented already in children and adolescents. The aim of this study was to examine CVD risk indicators and their determinants in healthy Swedish adolescents. The study population comprised 14- and 17-year-old boys and girls (n=1032), in the dty and surroundings of Umeå in northern Sweden. Biochemical, anthropometric, and physiological parameters associated to CVD (s- lipoproteins and s-apolipoproteins, s-insulin, s-ferritin, anthropometric measurements, blood pressure, and physical fitness) were evaluated in relation to family history of CVD, weight and length at birth, infant feeding regimen, physical growth during infancy and childhood, current diet, physical activity, smoking, and educational level and occupation of the parents. The main findings of the study were that, on average, total serum cholesterol (TC) values in boys and girls were at the same level as reported from other European countries. A family history of CVD, short duration of breast feeding, low attained height during infancy and childhood, high body mass index (BMI), and low physical fitness were all associated with an unfavourable serum lipid profile. The findings also showed that features typical of the insulin resistance syndrome are present already in adolescents. In boys, iron stores, estimated by serum ferritin, were related to BMI and physical fitness, in a similar way as well established CVD risk indicators. Compared to previous dietary studies in Sweden, mean relative (energy %) fat intake had decreased substantially although the mean relative intake of saturated fat was still rather high. For both boys and girls, reported relative energy intake (energy intake/estimated energy expenditure) decreased with increasing level of BMI. Furthermore, daily smoking was more common among adolescents from families with low socio-economic status (SES) but was most strongly associated to smoking in peers. Tobacco use was considerably higher among adolescents attending vocational programs at secondary high school as compared to theoretical programs. Daily smokers had a more unfavourable serum lipid profile compared to non-smokers. Low socio-economic status of the parents was related to higher BMI and low educational level to higher dietary fat intake in both boys and girls. In conclusion, the findings of the study show that parameters linked to adult CVD when examined in adolescents, are related to family history, infant nutrition, previous physical growth, current body composition, physical fitness, physical activity, smoking, and social status and educational level of the parents. / digitalisering@umu

Cardiovascular disease (CVD) is one of the major health issues of our time. The prevalence of CVD is increasing, both in industrialized and in developing countries, and causes suffering and a decreased quality of life for millions of people worldwide. CVD can have multiple etiologies, but the main underlying cause is atherosclerosis, which causes blood clot formation and obstructs vital arteries. Multiple risk factors of atherosclerosis have been identified, and body fatness is one of the most important ones.  The main aims of this thesis were to investigate the relation between body fatness and: CVD risk factors (paper I), incident stroke (paper II), and overall mortality (paper III). The results showed that abdominal obesity is strongly associated with both CVD risk factors and stroke incidence (papers I-II). The results also suggested that a substantial part of the association between increased body fat and stroke can be explained by an increase in traditional stroke risk factors associated with increased body fat (paper II). A gynoid fat distribution, with a high share of fat located around the hip, is, on the other hand, associated with lower risk factor levels in both men and women, and with a decreased risk of stroke in women (papers I-II). This illustrates the importance of assessing the overall distribution of body fat rather, than solely focusing on total body fatness. In elderly women, total body fat was found to be associated with increased survival, while abdominal fat moderately increased mortality risk (paper III). Lean mass (fat-free mass) was strongly associated with increased survival among elderly men and women (paper III). Erythrocyte sedimentation rate (ESR) is an indicator of inflammation and, possibly, an indicator of atherosclerotic disease. In paper IV, the relationship between ESR in young adulthood and the later risk of myocardial infarction (MI) was studied. Results showed that higher levels of ESR were associated with a higher MI risk, in a dose-responsive manner, and was independent of other well-established risk factors. In summary, both total and regional fat distribution are associated with CVD risk factors and stroke, but do not seem to correspond to an increase in mortality risk among the elderly. Also, inflammation, detected as an increase in ESR, is associated with long term MI risk in young men.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-14T13:30:00Z No. of bitstreams: 1 arquivo total.pdf: 2409615 bytes, checksum: 9c40d3feb8a769512e37ed55ee2c7c8e (MD5) / Made available in DSpace on 2017-02-14T13:30:00Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 2409615 bytes, checksum: 9c40d3feb8a769512e37ed55ee2c7c8e (MD5) Previous issue date: 2015-02-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Although clinical manifestations of cardiovascular disease (CVD) are observed, usually in the adult stage of life, there is strong evidence that these disorders can begin in adolescence. Few people who get a moderate to high risk for CVD at this stage can reverse this situation as an adult, which makes the monitoring of risk factors important element for detecting predisposition to the development of CVD in this population. Dietary modifications should form the basis for prevention of CVD, may reflect favorable changes in measurable risk factors. Dietary indices, such as the Healthy Eating Index (HEI) has been valid strategies for monitoring the power of a given population, and verification of cause and effect relationships in epidemiological studies. In Brazil, the HEI was adapted creating the Diet Quality Index Revised (DQI-R). This study aimed to analyze the relationship between diet quality and risk factors for CVD among adolescent students. Cross-sectional study with adolescents (N = 1077), between 10 and 12 years, of both sexes, enrolled in the sixth year of public schools in João Pessoa, Paraíba, Brazil. Risk factors for CVD were assessed: physical inactivity, sedentary behavior, increased waist circumference, overweight and high blood pressure. Food consumption was obtained using two recalls 24 h (R24h), being measured on a sub-sample. To evaluate the diet quality was used IQD-R. Ordinal logistic regression was used to identify the variables associated with HEI-R, (p <0.005). Less time in sedentary behavior (OR = 1.39, CI = 1.03 to 1.87%), as well as maternal education (OR = 0.55, 95% CI 0.38 to 0.81) were associated with higher IQD-R scores There was inadequate consumption of "whole fruit" (1.2 points, SD = 2.1), "whole grains" (1.3 points, SD = 1.6) and "dairy products" (2 0 points, SD = 3.0) and food represented by the "solid fat and added sugar and alcohol" (7.9 points, SD = 6.3). It emphasizes the importance of guidance for the reduction of sedentary behavior and actions to encourage the practice of healthy eating among adolescents. / Embora as manifestações clínicas das Doenças Cardiovasculares (DCV) sejam observadas, normalmente, na fase adulta da vida, há fortes evidências de que essas doenças podem ter início na adolescência. Poucas pessoas que adquirem um moderado a elevado risco para DCV nesta fase conseguem reverter esse quadro quando adulto, o que torna a monitorização dos fatores de risco elemento importante para detecção da predisposição ao desenvolvimento de DCV nesta população. Modificações dietéticas devem formar a base para a prevenção de DCV, podendo refletir em mudanças favoráveis nos fatores de risco mensuráveis. Os índices dietéticos, como o Healthy Eating Index (HEI), tem sido estratégias válidas no monitoramento da alimentação de uma determinada população, e na verificação de relações de causa e efeito em estudos epidemiológicos. No Brasil, o HEI foi adaptado criando-se o Índice de Qualidade da dieta Revisado (IQD-R). O presente estudo teve como objetivo analisar as relações entre qualidade da dieta e fatores de risco para DCV em adolescentes escolares. Estudo transversal realizado com adolescentes (N=1077), entre 10 e 12 anos, de ambos os sexos, matriculados no sexto ano de escolas públicas em João Pessoa, Paraíba, Brasil. Os fatores de risco para DCV avaliados foram: inatividade física, comportamento sedentário, circunferência da cintura aumentada, excesso de peso e pressão arterial elevada. O consumo alimentar foi obtido utilizando-se dois Recordatórios de 24 h (R24h), sendo um mensurado em uma subamostra. Para avaliação da qualidade da dieta foi utilizado o IQD-R. Regressão logística ordinal foi usada para identificação das variáveis associadas ao IQD-R, (p<0,005). Menor tempo em comportamento sedentário (OR=1,39; IC%= 1,03-1,87), além da escolaridade materna (OR=0,55; IC95%=0,38-0,81) foram associados a maiores pontuações do IQDR Houve consumo inadequado de “frutas integrais” (1,2 pontos; DP= 2,1), “cereais integrais” (1,3 pontos; DP= 1,6) e “leite e derivados” (2,0 pontos; DP=3,0) e de alimentos representados pelo grupo “gordura sólida e açúcar de adição e álcool” (7,9 pontos; DP=6,3). Ressalta-se a importância da orientação em relação à redução do comportamento sedentário e ações de incentivo à prática de alimentação saudável entre adolescentes.