The effects of melatonin supplementation on vascular tissue during first line ART: an in vivo, ex vivo and in vitro study

Rawstorne, Jordyn (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

Introduction: Although Antiretroviral therapy (ART) has dramatically reduced HIVassociated morbidity and mortality, non-HIV-related conditions and comorbidities continue to rise in this population. Cardiovascular disease (CVD) has been reported to be the leading cause of death in the HIV-positive population receiving ART. ART is thought to impair vascular endothelial function through increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. In this study, we aim to assess the effects of melatonin - a potent antioxidant -supplementation during ART on specific intracellular products of rat Aortic Endothelial Cells (AECs) as well as on the vascular reactivity of rat aortas. Methods: Cells were serum starved and treated with three different melatonin (1nM; 1uM; 10uM) or ART (Low: EFV: 5μM; FTC: 5μM; TDF: 80nM; Mid: EFV: 8μM; FTC: 7.5μM; TDF: 400nM; High: EFV: 12μM; FTC: 10μM; TDF: 1μM) concntrations for up to 24 hours. Nitric oxide (NO), RNS and necrosis were measured with a platereader (control expressed as 100%). The concentration that resulted in the greatest differences, compared to untreated cells, was selected for co-treatment studies and protein investigations, where the same parameters were measured. The effects of acute melatonin and ART administration on vascular reactivity was measured by aortic ring isometric tension studies in aortas extracted from control male Wistar rats. The endothelium-dependent and independent vascular reactivity was measured by isometric tension studies on aortas harvested from male Wistar rats that were treated for 8 weeks with melatonin (10mg/kg/day) and/or ART (EFV: 51.6 mg/kg; FTC: 17.4 mg/kg; TDF: 25.8 mg/kg). Signalling proteins involved in these changes were measured by western blot analyses. Results (Mean±SEM): Dose Response Studies: 1nM melatonin decreased necrosis [92.56±3.11%;p=0.0004] compared to untreated controls [100.00±1.04%; p=0.0004]. Highconcentration ART lead to increased NO production [112.70±2.17%; Control:100.00±2.22%;p=0.0015], RNS production [108.80±2.20%; Control:100±0.79%;p=0.0152] and necrosis [107.30±1.34%; Control: 100.00±0.86%;p=0.0251], compared to untreated controls. Main Studies: 1nM melatonin decreased necrosis [92.43±3.75%;p=0.0002], while Highconcentration ART increased necrosis [121.30±9.11%;p=0.0002] compared to untreated cells [100.00±1.07%;p=0.0002]. When combined, 1nM melatonin + High-concentration ART decreased necrosis [94.17±5.08%;p=0.0002] compared to ART alone. Western blot analyses (arbitarty units) showed that ART increased nitrotyrosine levels [2.31±0.34; Control:1.00±0.18;p=0.0486], but decreased p22 PHOX [0.20±0.043; Control:1.00±0.15;p<0.0001], and cleaved caspase-3 [0.25±0.038; Control:1.00±0.18;p=0.0005], expression. In actute aortic ring experiments, ART exposure elicited a burst of contraction during the treatment period, followed by a significant attenuation in accumulative contraction compared to all other groups. In endothelium-dependent and independent contraction studies on aortas from treated rats, all groups showed a pro-contractile response compared to the control. Western blot analyses showed that ART decreased cleaved caspase-3 [0.27±0.08;p=0.0055] expression. Conclusion: Decreased necrosis in AECs treated with combined melatonin and ART, compared to AECs treated with ART alone shows the protective effect of melatonin. Further specific protein investigations are needed to elucidate this mechanism. Western blots showed that ART induced anti-apoptotic effects and increased RNS production, but not NADPH-oxidase activity. The initial contractile burst following acute ART exposure may precondiition the aortas, resulting in the decreased accumulatve contractile capacity. Chronic ART treatment studies showed that ART treatment does not seem to affect vasorelxation. Blot data reconfirmed that ART is also anti-apoptoic in vivo.

Inleiding: Alhoewel antiretrovirale terapie HIV verwante morbiditeit en mortaliteit dramaties verminder het, styg nie-HIV-verwante toestande in hierdie populasie. Kardiovaskulêre siektes word beskou as die hoof oorsaak van sterftes in die HIV positiewe populasie wat antiretrovirale terapie (ART) ontvang. ART kan die vaskulêre endoteelfunksie benadeel deur verhoogde reaktiewe suurstof species (ROS) en reaktiewe stikstof species (RNS) produksie. In hierdie studie was die effekte van melatonien (kragtige antioksidant) aanvulling gedurende ART ondersoek, met spesifieke verwysing na intrasellulêre afskeidingsprodukte van rot aorta endoteelselle, asook in die konteks van rot aorta vaskulêre reaktiwiteit. Metodes: Selle was onderwerp aan serum weerhouding en behandel met drie verskillende melatonien (1nM; 1uM; 10uM) of ART (Laag: EFV: 5μM; FTC: 5μM; TDF: 80nM; Medium: EFV: 8μM; FTC: 7.5μM; TDF: 400nM; Hoog: EFV: 12μM; FTC: 10μM; TDF: 1μM) konsentrasies vir tot 24uur. Stikstofoksied (NO), RNS and nekrose is gemeet met ‘n plaatleser (kontrole uitgedruk as 100%). Die konsentrasie wat gelei het tot die grootste verskille, in vergelyking met die onbehandelde selle, was gekies vir kobehandeling studies en proteïen ontledings, waar dieselfde veranderlikes gemeet was. Die effekte van akute melatonien en ART toediening op vaskulêre reaktiviteit is gemeet d.m.v. aortiese ring isometriese spanningstudies in aortas afkomstig van gesonde manlike Wistar rotte. Die endoteel-afhanklike en onafhanklike vaskulêre reaktiwiteit is gemeet d.m.v. isometriese spanningstudies in aortas afkomstig van manlike Wistar rotte wat vir 8 weke behandel was met melatonien (10mg/kg/day) en/of ART (EFV: 51.6 mg/kg; FTC: 17.4 mg/kg; TDF: 25.8 mg/kg). Seinproteïne in hierdie weefsel is gemeet deur Western blot analises. Resultate (Mean±SEM): Dosis-reaksie Ondersoeke: 1nM melatonien het nekrose [92.56±3.11%;p=0.0004] laat verminder in vergelyking met onbehandelde kontrole [100.00±1.04%;p=0.0004]. Hoë-konsentrasie ART het tot verhoogde NO produksie [112.70±2.17%; kontrole:100.00±2.22%;p=0.0015], RNS produksie [108.80±2.20%; kontrole:100.00±0.79%;p=0.0152] en nekrose [107.30±1.34%; kontrole: 100.00±0.86%; p=0.0251] gelei, in vergelyking met onbehandelde kontrole. Hoof Ondersoeke: 1nM melatonien nekrose [92.43±3.75%;p=0.0002] laat toeneem, terwyl hoë-konsentrasie ART nekrose 121.30±9.11%;p=0.0002] in vergelyking met onbehandelde selle [100.00±1.07%;p=0.0002] verhoog. Tydens gekombineerde behandeling, het 1nM melatonien + Hoë-konsentrasie ART nekrose [94.17±5.08%; p=0.0002] verlaag teenoor ART alleen. Western blot analises (arbitrêre eenhede) het getoon dat ART nitrotirosien vlakke vermeerder het [2.31±0.34; kontrole:1.000±0.18;p=0.0486], maar p22 PHOX [0.20±0.04; kontrole:1.00±0.15;p<0.0001], en gekliefde kaspase-3 [0.25±0.038; kontrole:1.00±0.18;p=0.0005] uitdrukking verlaag het. In akute aorta ring eksperimente, het ART blootstelling ‘n skielike en kortstondige kontraksie gedurende die behandelings periode veroorsaak, gevolg deur ‘n beduidende verswakking in akkumulatiewe kontraksie teenoor al die ander groepe. In endoteel-afhanklike en onafhanklike kontraksie studies op aortas van behandelde rotte, het alle groepe ‘n pro-kontraktiele reaksie getoon in vergelyking met die kontrole. Western blot analises het getoon dat ART v gekliefde kaspase-3 uitdrukking verlaag het [0.27±0.08;p=0.0055]. Gevolgtrekking: Die verlaagde nekrose wat in aorta endoteelselle (AECs), behandel met gekombineerde melatonien en ART, waargeneem is teenoor AECs wat slegs ART ontvang het, is ‘n bewys van die beskermde effekte van melatonien. Toekomstige studies wat spesifieke proteïene ondersoek is nodig om die meganisme verder te verklaar. Western blots ondersoeke het gewys dat ART anti-apoptotiese effekte en vermeerde RNS produksie veroorsaak, maar geen effekte op NADPH-oksidase aktiwiteit toon nie. Die aanvanklike kontraktiele respons tydens akute ART blootstelling kan die aortas vooraf kondisioneer wat ‘n verlaagde akkumulatiewe kontraktiele kapasiteit veroorsaak het. Chroniese ART behandeling studies het getoon dat die behandeling nie die bloedvatverslapping beïnvloed nie. Die Western blot resultate van die in vivo studies het die anti-apoptotiese effekte van ART herbevestig.

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