Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research Foundation Introduction Endothelial dysfunction (ED) is an early precursor of cardiovascular disease characterized by decreased nitric oxide (NO) and increased oxidative stress, creating a pro-inflammatory environment. Increased ED and cardiovascular risk have been observed in HIV-1 infection and antiretroviral therapy (ART). Purpose The United Nations AIDS update indicates that in 2015 17.0 million people worldwide were living with HIV and receiving antiretroviral therapy. Considering the extent of this global pandemic and its comorbidity with CVD, zooming in on HIV’s intersection with antiretroviral therapy and cardiovascular health is of paramount importance. Yet, many of the mechanisms involved are still poorly understood. This knowledge can ultimately assist with the careful selection of antiretroviral-agents in patients with underlying cardiovascular risk factors. Objectives Establishing a simulated model of in vitro HIV-1-infection in aortic endothelial cells and determining the effects of non/nucleoside reverse transcriptase inhibitors (NRTI/NNRTIs) and protease inhibitors (PIs) on markers of endothelial function and the expression/activation of vascular signalling proteins. Methods A simulated HIV-1-model was established by adding recombinant HIV-1 proteins to growth medium in a single cocktail containing Tat, Gp160 and Nef. ART1 (NRTI/NNRTIs) were administered as a single cocktail to growth medium, containing efavirenz (EFV), emtricitabine (FTC) and tenofovir (TDF). ART2 (PIs) were administered as a single cocktail to growth medium, containing lopinavir (LPV) and ritonavir (RTV). All the aforementioned treatments were subject to dose-response investigations and treatment lasted 24 hours. Cell viability, NO-production and oxidative stress were measured using fluorescent probes. Selected dosages from above studies were further used and important vascular signalling proteins in HIV-1 exposed AECs were investigated by western blotting. Results HIV-1 (100ng/ml, 24h) resulted in ↓NO-levels (DAF-fluorescence intensity: 100±1.25% vs. 76.00±3.67%; p<0.05) with no oxidative stress. HIV-1+PI treatment lowered NO levels (DAF-fluorescence: 100±0.72% vs. 94.47±1.91%; p<0.05). HIV-1+ NRTI/NNRTI treatment had no effects. HIV-1+PI treatment ↓IκBα expression (inhibitory signalling protein in pro-inflammatory NF-κB pathway) (1.0 ± 0.15 vs 0.37 ± 0.03; p<0.05) and ↓eNOS expression (1.0 ± 0.21 vs. 0.28 ± 0.04; p<0.05). The same was observed for PKB/Akt (1.0 ± 0.03 vs. 0.68 ± 0.02; p<0.05). No effects on cell viability and oxidative stress were observed. Conclusion Exposure of AECs to combined HIV-1-protein and PI treatment lowered NO-production, which may be linked to underlying PKB/Akt and eNOS downregulation. HIV-1+PI treatment was associated with increased pro-inflammatory NF-κB signalling. Overall, our results suggest that a dysfunctional state was induced in AECs exposed to HIV-1 proteins and PI treatment.

中文翻译：

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HIV-1 蛋白和抗逆转录病毒治疗对主动脉内皮细胞 (AEC) 的影响。机械体外方法


资金致谢 资金来源类型： 公共补助金 – 仅国家预算。主要资金来源：国家研究基金会 简介 内皮功能障碍 (ED) 是心血管疾病的早期先兆，其特征是一氧化氮 (NO) 减少和氧化应激增加，从而产生促炎环境。在 HIV-1 感染和抗逆转录病毒治疗 (ART) 中观察到 ED 和心血管风险增加。目的 联合国艾滋病最新情况表明，2015 年全世界有 1700 万人感染艾滋病毒并接受抗逆转录病毒治疗。考虑到这一全球流行病的严重程度及其与心血管疾病的合并症，重点关注艾滋病毒与抗逆转录病毒治疗和心血管健康的交叉点至关重要。然而，许多涉及的机制仍然知之甚少。这些知识最终可以帮助有潜在心血管危险因素的患者仔细选择抗逆转录病毒药物。目的 建立主动脉内皮细胞体外 HIV-1 感染模拟模型，确定非/核苷类逆转录酶抑制剂 (NRTI/NNRTI) 和蛋白酶抑制剂 (PI) 对内皮功能标志物和内皮细胞表达/激活的影响。血管信号蛋白。方法 通过将重组 HIV-1 蛋白添加到含有 Tat、Gp160 和 Nef 的单一混合物的生长培养基中，建立了模拟 HIV-1 模型。 ART1（NRTI/NNRTI）作为单一混合物施用到含有依非韦伦（EFV）、恩曲他滨（FTC）和替诺福韦（TDF）的生长培养基中。 ART2 (PI) 作为单一混合物施用到含有洛匹那韦 (LPV) 和利托那韦 (RTV) 的生长培养基中。 所有上述治疗均接受剂量反应研究，治疗持续24小时。使用荧光探针测量细胞活力、NO 产生和氧化应激。进一步使用上述研究中选定的剂量，并通过蛋白质印迹研究了 HIV-1 暴露的 AEC 中重要的血管信号蛋白。结果 HIV-1（100ng/ml，24 小时）导致 ↓NO 水平（DAF 荧光强度：100±1.25% 对比 76.00±3.67%；p<0.05），无氧化应激。 HIV-1+PI治疗降低了NO水平（DAF荧光：100±0.72% vs. 94.47±1.91%；p<0.05）。 HIV-1+ NRTI/NNRTI 治疗没有效果。 HIV-1+PI 治疗 ↓IκBα 表达（促炎 NF-κB 通路中的抑制性信号蛋白）（1.0 ± 0.15 对比 0.37 ± 0.03；p<0.05）和 ↓eNOS 表达（1.0 ± 0.21 对比 0.28 ± 0.04；p <0.05）。 PKB/Akt 也观察到同样的情况（1.0 ± 0.03 对比 0.68 ± 0.02；p<0.05）。没有观察到对细胞活力和氧化应激的影响。结论 AEC 接受 HIV-1 蛋白和 PI 联合治疗可降低 NO 产生，这可能与潜在的 PKB/Akt 和 eNOS 下调有关。 HIV-1+PI 治疗与促炎 NF-κB 信号传导增强相关。总体而言，我们的结果表明，暴露于 HIV-1 蛋白和 PI 治疗的 AEC 中诱导了功能障碍状态。