The Effects of HIV-1-proteins and Antiretroviral Therapy on Aortic Endothelial Cells (AECs) – A Mechanistic in vitro Approach

Marincowitz, Clara Maria Elizabeth (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

Introduction: Endothelial dysfunction is an early precursor of cardiovascular disease characterized by decreased nitric oxide (NO) levels following the development of oxidative stress. Oxidative stress has been shown to result not only in the inactivation of NO itself, but also of endothelial NO synthase (eNOS), the enzyme responsible for NO synthesis. This contributes to creating a pro-inflammatory environment in the vasculature, which can lead to atherosclerosis and cardiovascular disease. Increased endothelial dysfunction and cardiovascular risk have been observed in both HIV-1 infection and antiretroviral therapy (ART). Objectives: To establish a simulated model of in vitro HIV-1-infection and determine the effects of non/nucleoside reverse transcriptase inhibitors (NRTI/NNRTIs) and protease inhibitors (PIs) on markers of endothelial function and the expression/activation of important vascular signalling proteins within this HIV-1-model. Methods: A simulated HIV-1-model was established by adding recombinant HIV-1 proteins (100 ng/ml each of Nef, Tat and Gp160) to the growth medium of cultured rat aortic endothelial cells (AECs). Subsequently, the effects of NRTI/NNRTIs (efavirenz, emtricitabine and tenofovir) and PIs (lopinavir and ritonavir) on these HIV-1 exposed AECs were determined using fluorescent probes to assess cell viability, NO-production and oxidative stress via reactive nitrogen species (RNS) production. The expression/activation of important vascular signalling proteins, including eNOS and IκBα (an inhibitor of the inflammatory NFκB signalling pathway), was also evaluated by western blotting. Results: Exposure to 100 ng/ml of HIV-1 Nef, Tat and Gp160 for 24 hours led to a significant decrease in NO production in AECs (DAF-2/DA fluorescence intensity: 72.05±8.37% vs. 100±1.55%). NRTI/NNRTI treatment within an HIV-1-protein medium environment for 24 hours had no effect on NO production, possibly abrogating the reduced levels observed with HIV-1-protein treatment on its own. Furthermore, a decrease in RNS was observed (DHR-123 fluorescence intensity: 83.19±3.5% vs. 100±0.22%). PI treatment within an HIV-1-protein medium environment for 24 hours resulted in a reduction in NO production in a concentration-dependent manner (DAF-2/DA fluorescence intensity: 92.74±1.4% vs. 100±0.67% and 85±1.81% vs. 100±0.56%), probably due to decreased eNOS expression (0.28±0.04 vs. 1±0.21). Interestingly, neither HIV-1 protein exposure on its own, nor PI treatment in isolation had any effects on eNOS. The same was observed for IκBα, where combined HIV-1-protein and PI exposure reduced levels (0.37±0.03 vs. 1±0.15) and neither of these treatments in isolation had any effects. Conclusion: HIV-1 Nef, Tat and Gp160 attenuated NO production in AECs, while NRTI/NNRTI treatment within this HIV-1 protein environment showed minimal adverse effects, and could possibly even be beneficial, potentially reversing the detrimental consequences of HIV-1 proteins on NO production. PI treatment, on the other hand, seemed to demonstrate a harmful interaction with HIV-1 proteins, resulting in a downregulation of the eNOS-NO biosynthesis pathway. The PI-HIV-1 protein combination was also associated with up-regulation of the pro-inflammatory NFκB signalling pathway, which may provide an explanation for the decreased eNOS expression.

Inleiding: Endoteeldisfunksie is ‟n voorloper van kardiovaskulêre siektes wat gepaard gaan met verlaagde stikstofoksied (NO) vlakke as gevolg van oksidatiewe stres. Studies het getoon dat oksidatiewe stres nie net NO inaktiveer nie, maar ook endoteel NO sintase (eNOS), die ensiem verantwoordelik vir NO sintese. Hierdie dra by tot die ontwikkeling van ‟n pro-inflammatoriese toestand in die vaskulêre omgewing, wat kan lei tot aterosklerose en kardiovaskulêre siektes. Verhoogde endoteeldisfunksie en kardiovaskulêre risiko is al in beide HIV-1-infeksie en antiretrovirale terapie gemerk. Doelstellings: Om ‟n gesimuleerde model van in vitro HIV-1-infeksie te ontwikkel en vas te stel wat die gevolge van behandeling met nie-/nukleosied tru-transkriptase inhibeerders (NRTI/NNRTIs) en protease inhibeerders (PIs) op merkers van endoteeldisfunksie en die uitdrukking/aktivering van belangrike vaskulêre seinproteïne in hierdie HIV-1-model is. Metodes: ‟n Gesimuleerde HIV-1-model is ontwikkel deur rekombinante HIV-1 proteïne (100 ng/ml elk van Nef, Tat en Gp160) by die groei media van gekweekte rot aorta endoteelselle (AESe) te voeg. Hierna is die uitwerking van NRTI/NNRTIs (efavirenz, emtricitabine en tenofovir) en PIs (lopinavir en ritonavir) op die HIV-1 blootgestelde AESe vasgestel met die gebruik van fluoressensie ondersoeke om sel lewensvatbaarheid, NO-produksie en oksidatiewe stres via die produksie van reaktiewe stikstof spesies (RSS) te assesseer. Die uitdrukking/aktivering van belangrike vaskulêre seinproteïne, insluitend eNOS en IκBα (‟n onderdrukker van die inflammatoriese NFκB seinpaaie), is geëvalueer deur middel van western blot analises. Resultate: Blootstelling aan 100 ng/ml elk van HIV-1 Nef, Tat en Gp160 vir 24 uur het gelei tot ‟n beduidende vermindering in NO produksie in AESe (DAF-2/DA fluoressensie intensiteit: 72.05±8.37% vs. 100±1.55%). NRTI/NNRTI behandeling in ‟n HIV-1-protein medium omgewing vir 24 uur het geen uitwerking op NO produksie gehad nie en het moontlik die verminderde vlakke weens HIV-1-protein behandeling op sy eie verbeter. Bowendien, is ‟n afname in RSS gemerk (DHR-123 fluoressensie intensiteit: 83.19±3.5% vs. 100±0.22%). PI behandeling in ‟n HIV-1-protein media omgewing vir 24 uur het verminderde NO produksie tot gevolg gehad in ‟n dosis-afhanklike wyse (DAF-2/DA fluoressensie intensiteit: 92.74±1.4% vs. 100±0.67% en 85±1.81% vs. 100±0.56%), waarskynlik as gevolg van verminderde eNOS uitdrukking (0.28±0.04 vs. 1±0.21). Interessant genoeg, het nóg HIV-1 proteïne, nóg PI behandeling op hul eie enige uitwerking op eNOS gehad nie. Dieselfde is waargeneem vir IκBα, waar blootstelling aan gekombineerde HIV-1-protein en PI behandeling IκBα vlakke verlaag het (0.37±0.03 vs. 1±0.15), terwyl die HIV-1 en PI behandelings op hul eie geen uitwerking gehad het nie. Gevolgtrekking: HIV-1 Nef, Tat en Gp160 het NO produksie in AESe verminder, terwyl NRTI/NNRTI behandeling binne hierdie HIV-1 proteïen omgewing minimale negatiewe gevolge getoon het en moontlik selfs voordelig was, deur potensieel die negatiewe uitwerkings van HIV-1 proteïne op NO produksie te herstel. PI behandeling, aan die ander kant, het geblyk om ‟n skadelike interaksie met die HIV-1 proteïne te toon, wat gelei het tot ‟n afname in eNOS-NO biosintese. Die PI-HIV-1 proteïen kombinasie was ook geassosieer met ‟n opregulering van die pro-inflammatoriese NFκB seinpaaie, wat moontlik ‟n verklaring is vir die verminderde eNOS uitdrukking.

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