| Summary: | Thesis (MScMedSc)--Stellenbosch University, 2015. === ENGLISH ABSTRACT: Ataxia telangiectasia (AT) is a well-characterized neurodegenerative disease resulting from a genetic defect in the Atm gene causing an absence or very low expression of the ATM protein. As AT patients are prone to the development of insulin resistance and atherosclerosis, the aim or the current study was to investigate the importance of the ATM protein in the endothelium and its role in the signalling pathways of nitric oxide (NO) production. To accomplish this, the first objective was to establish an in-house endothelial cell isolation technique harvested from normal and insulin resistant animals. Unfortunately, these cultures, although staining positive with an endothelial cell specific stain, were not pure enough and did not express endothelial NO synthase (eNOS), the central enzyme in NO production.
The remainder of the study utilized commercial aortic endothelial cells (AECs) and found that there was a significant increase in NO production when the ATM protein was inhibited by the specific inhibitor, Ku-60019. The beneficial impact of increased NO production includes maintaining vascular homeostasis, promoting angiogenesis, initiating DNA repair by activating p53 and inhibiting smooth muscle cell proliferation. On the other hand, reactive oxygen species (ROS) and reactive nitrogen species (RNS) also generated by high levels of NO, can exert both protective and harmful effects. Examples of these include cell death due to high concentrations of ROS. However, Ku-60019 did not result in increased cell death of AECs.
We demonstrated for the first time, a relationship between endothelial ATM protein kinase and the generation of NO. The signalling pathways involved in NO production and glucose utilization form a network of interrelationships. Central to both pathways is the activity of two protein kinases, PKB/Akt and AMPK. Both these kinases are known to phosphorylate the eNOS enzyme to produce NO on the one hand and AS160 to induce GLUT 4 translocation and glucose uptake on the other hand. Activation of the ATM protein is postulated to be a prerequisite for PKB/Akt activation and it may also result in activation of AMPK. However, using insulin to stimulate ATM, we could not show that inhibition of ATM in endothelial cells affected expression or insulin-stimulated activation of PKB/Akt while the PI3-K inhibitor wortmannin, inhibited the latter. In addition, inhibition of ATM negatively regulated the phospho/total ratio of AMPK. We therefore postulate that the NO production elicited by inhibition of ATM, may not be as result of eNOS activity.
A second important observation was that inhibition of ATM significantly enhanced phosphorylation of the p85 regulatory subunit of PI3-K. This would imply that ATM normally has an inhibitory effect on p85 phosphorylation and therefore PI3-K activation. We base this assumption on previous publications showing that Ku-60019 does not inhibit PI3K. This again indicates that ATM has a hitherto unexplored regulatory role in endothelial function. === AFRIKAANSE OPSOMMING: Ataxia telangiectasia (AT) is a goed-gekarakteriseerde neurodegeneratiewe siekte a.g.v. ‘n genetiese afwyking in the Atm geen wat lei tot ‘n afwesige of lae uitdrukking van die ATM proteïen. Aangesien AT pasiënte geneig is om insulienweerstandigheid en aterosklerose te ontwikkel, was die doel van hierdie studie om die belang van die ATM proteïen in die endoteel, en sy rol in die seintransduksiepaaie betrokke by stikstofoksied (NO) produksie, te ondersoek. Om dit te bereik, was die eerste mikpunt om ‘n eie endoteelsel isolasie-tegniek (ge-oes van normale en insulienweerstandige diere) te vestig. Ongelukkig was hierdie selkulture nie suiwer genoeg nie.Ten spyte daarvan dat hulle positief getoets het met ‘n endoteelsel-spesifieke kleurstof kon geen uitdrukking van eNOS, die sentrale ensiem verantwoordelik vir NO produksie, waargeneem word nie.
Die res van die studie het van kommersiële aorta endoteelselle (AES) gebruik gemaak, en daar is gevind dat die inhibisie van die ATM proteïen met die spesifieke inhibitor, Ku-60019, tot ‘n beduidende toename in NO produksie gelei het. Die voordelige impak van verhoogde NO produksie sluit die handhawing van vaskulêre homeostase, bevordering van angiogenese, inisiëring van DNA herstel deur p53 aktivering en inhibisie van gladdespiersel proliferasie in. Reaktiewe suurstofspesies (ROS) en reaktiewe stikstofspesies (RNS) wat ook a.g.v.verhoogde NO gegenereer word, kan egter beide beskermende sowel as skadelike effekte uitoefen. Voorbeelde sluit seldood a.g.v. hoë ROS konsentrasies in. Ku-60019 het egter nie tot ‘n toename in seldood van die AES gelei nie.
Hierdie studie het vir die eerste keer aangetoon dat daar ‘n verwantskap tussen die endoteel ATM proteïen kinase en die produksie van NO bestaan. Die seintransduksie paaie betrokke by NO produksie en glukose verbruik vorm ‘n interafhanklike netwerk. Die aktiwiteit van twee proteïen kinases, PKB/Akt en AMPK, is sentrale rolspelers in beide paaie. Albei hierdie kinases is daarvoor bekend dat hulle die eNOS ensiem fosforileer om NO te produseer, maar terselfdertyd ook lei tot AS160 fosforilering, wat tot GLUT 4 translokering en glukose opname lei. Dis is voorgestel dat aktivering van die ATM proteïen ‘n voorvereiste vir PKB/Akt aktivering mag wees en verder kan dit ook tot aktivering van AMPK lei. Ons kon nie aantoon dat inhibisie van ATM in endoteelselle die uitdrukking of insulien-geïnduseerde aktivering van PKB/Akt onderdruk nie, terwyl die PI3-K inhibitor, wortmannin, wel laasgenoemde geïnhibeer het. Verder het die inhibisie van ATM die fosfo/totale AMPK verhouding negatief gereguleer. Ons postuleer dus dat die NO produksie waargeneem tydens ATM inhibisie, moontlik nie die gevolg van eNOS aktiwiteit was nie.
‘n Tweede belangrike waarneming was dat die inhibisie van ATM die fosforilering van die p85 regulatoriese subeenheid van PI3-K beduidend laat toeneem het. Dit impliseer dat ATM normaalweg ‘n inhibitoriese effek op p85 fosforilering, en dus PI3-K aktivering, het. Hierdie aanname word gemaak n.a.v. vorige publikasies wat getoon het dat Ku-60019 nie PI3-K inhibeer nie. Dit dui weer eens daarop dat ATM ‘n tot nog toe onbekende regulatoriese rol in endoteelfunksie het.
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