Thesis (DScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Vascular endothelium forms the first line of defence against harmful stimuli in the circulation.
Endothelial dysfunction is a valuable predictor of cardiovascular disease and therapies aimed at
improving endothelial function are therefore needed. The anti-dyslipidaemic agents, simvastatin
and fenofibrate, are known for their beneficial effects on lipid parameters, however additional
pleiotropic effects have been shown for both. These include improved endothelial function due
to increased levels of nitric oxide (NO), as well as anti-oxidant and anti-inflammatory actions. NO
is produced by the enzyme, nitric oxide synthase (NOS), which exists in the endothelial NOS
(eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) isoforms. Most studies investigating the
endothelial effects of simvastatin and fenofibrate are performed on macrovascular-derived
endothelial cells, and there is a lack of data on endothelial cells (ECs) from the microcirculation,
particularly the cardiac microvessels.
This dissertation aimed to investigate and elucidate mechanisms underlying the pleiotropic
effects of simvastatin and fenofibrate on ECs and vascular tissue using in vitro, ex vivo and in vivo
experimental models. In vitro investigations included flow cytometry-based intracellular
measurements of NO, as well as different types of reactive oxygen species (ROS) and cell viability
parameters. Signalling pathways involved with these changes were measured by western blot
analyses of the expression and phosphorylation of critical proteins involved in vascular function.
Results on cardiac microvascular ECs (CMECs) demonstrated that fenofibrate (50 μM) exerted a
potent, increasing effect on NO production after short periods (1 and 4 hour treatments), but
after 24 hours the effects were less robust. Exhaustive investigations suggested that the NOincreasing
effects of fenofibrate in baseline CMECs were NOS-independent, a novel finding as far
as we are aware. Fenofibrate’s ability to protect ECs against injury was demonstrated when
CMECs incubated with the pro-inflammatory cytokine, TNF-α, were pre-treated with fenofibrate,
resulting in increased NO and improved cell viability parameters. Simvastatin (1 μM) increased
NO to a lesser extent in baseline CMECs, and resulted in increased apoptosis and necrosis.
Following the cell studies, their effects on vascular reactivity was measured by aortic ring
isometric tension studies. The effects of acutely administered fenofibrate to pre-contracted
aortic rings were investigated, and results showed a modest, but significant NOS-dependent
vasodilatory response. Next, an in vivo model of Wistar rats treated with simvastatin (0.5
mg/kg/day) and fenofibrate (100 mg/kg/day) for 6 weeks was established. Data showed that
neither drug was able to improve aortic ring contraction and dilation above baseline values. Both
drug treatments increased iNOS expression, which is usually associated with harmful actions.
However, in our hands, increased iNOS expression was associated with a beneficial anticontractile
response in the simvastatin-treated animals. Fenofibrate treatment increased NO
bioavailability in the blood of these animals.
In conclusion, fenofibrate showed endothelio-protective pleiotropic effects with regards to NO
production after short treatment periods in CMECs. These effects were mediated via a NOSindependent
mechanism, a novel finding. Fenofibrate pre-treatment was also protective against
the harmful effects of TNF-α. Simvastatin did not show pronounced pleiotropic effects in vitro
or in vivo on endothelial function. / AFRIKAANSE OPSOMMING: Die vaskulêre endoteellaag is die eerste linie van verdediging teen skadelike stimuli in die
bloedsirkulasie. Endoteeldisfunksie is ‘n waardevolle voorspeller van kardiovaskulêre siektes en
enige terapeutiese behandeling wat kan bydra tot verbeterde endoteelfunksie is belangrik.
Simvastatien en fenofibraat word as anti-dislipidemiese middels voorgeskryf en hoewel hulle
primêr gebruik word om cholesterolvlakke te verbeter, toon hulle ook pleiotropiese (cholesterolonafhanklike)
eienskappe. Dit sluit in bevordering van endoteelfunksie (via verhoogde
stikstofoksied (NO) produksie), asook anti-oksidant en anti-inflammatoriese effekte. NO word
vervaardig deur die ensiem, stikstofoksiedsintase (NOS) wat voorkom in drie isovorme: endoteelafgeleide
NOS (eNOS), induseerbare NOS (iNOS) en neuronale NOS (nNOS). Die meerderheid
studies wat pleiotropiese effekte van simvastatien en fenofibraat ondersoek, gebruik
endoteelselle van makrovaskulêre bloedvate, wat beteken daar is ‘n tekort aan data aangaande
endoteelselle vanaf mikrovaskulêre vate, veral kardiale mikrovaskulêre vate (CMECs).
Hierdie proefskrif het dit ten doel gehad om meganismes betrokke by die pleiotropiese effekte
van simvastatien en fenofibraat te ondersoek deur van in vitro, ex vivo en in vivo modelle gebruik
te maak. Die in vitro ondersoeke het gefokus op vloeisitometrie-gebaseerde metings van
intrasellulêre NO, reaktiewe suurstof-radikale (ROS) en sellewensvatbaarheid. Seintransduksie
paaie betrokke by hierdie veranderinge was bepaal deur proteienuitdrukking en -fosforilasie
vlakke te meet van belangrike proteïene, met behulp van die Western-blot tegniek.
Resultate van die CMEC eksperimente het getoon dat fenofibraat (50 μM) ‘n kragtige en
verhogende effek op NO produksie uitgeoefen het na kort behandelingstye (1 en 4 ure), maar na
24 uur was hierdie effek minder uitgesproke. Uitvoerige ondersoeke het getoon dat fenofibraat
se basislyn effekte op CMECs deur NOS-onafhanklike meganismes teweeggebring is, en sover ons
kennis strek, is dit ‘n nuwe bevinding. Fenofibraat se endoteel-beskermende effekte kon ook
aangetoon word deur CMECs vir een uur te behandel voor byvoeging van die pro-inflammatories
sitokien, tumor nekrose faktor alpha (TNF-α), wat gelei het tot verhoogde NO vlakke en
verbeterde seloorlewing. Simvastatien (1 μM) het tot ‘n mindere mate NO produksie verhoog in
CMECs, tesame met pro-apoptotiese en -nekrotiese effekte.
Vervolgens was die effekte op vaskulêre reaktiwiteit geëvalueer d.m.v. isometriese
spanningsondersoeke. Akute effekte van fenofibraat is gemeet deur byvoeging daarvan tot ‘n
vooraf saamgetrekte aorta-ring, wat tot matige, maar beduidende NOS-afhanklike verslapping
gelei het. Hierna is ‘n in vivo model opgestel deur Wistar rotte vir ses weke met 0.5 mg/kg/dag
simvastatien of 100 mg/kg/dag fenofibraat te behandel. Resultate toon dat geen van die
behandelings basislyn kontraksie of verslapping van aorta ringe kon verbeter nie. Beide
behandelings het tot verhoogde iNOS uitdrukking gelei, wat gewoonlik met nadelige effekte
geassosieer word, maar in ons studies was dit met voordelige, anti-kontraktiele effekte in aortaringe
van simvastatien-behandelde rotte geassosieer. Fenofibraat behandeling het die NObiobeskikbaarheid
in die rotte se bloed verhoog.
Ten slotte, fenofibraat het met endoteel-beskermende, pleiotropiese effekte op endoteelselle
gepaard gegaan, veral t.o.v. NO-produksie na akute middeltoediening in die CMECs. Die
meganisme was ‘n NOS-onafkanklike proses, wat ‘n nuwe bevinding is. Fenofibraat prebehandeling
het teen die skadelike effekte van TNF-α beskerm. Geen uitgesproke pleiotropiese
effekte is in vitro of in vivo gevind met simvastatien behandeling nie.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96651 |
| Date | 03 1900 |
| Creators | Westcott, Corli |
| Contributors | Strijdom, Hans, Huisamen, Barbara, Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xxxi, 333 pages : illustrations (some colour) |
| Rights | Stellenbosch University |
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