Abstract
BackgroundAspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, although specific mechanisms are poorly understood. Recently, PIs, particularly Nelfinavir, were reported to disrupt Notch signaling in the HIV-related endothelial cell neoplasm, Kaposi's sarcoma. Given the importance of maintaining proper cerebral endothelial cell signaling at the blood brain barrier during HIV infection, we considered potential signaling pathways such as Notch, that may be vulnerable to dysregulation during exposure to PI-based anti-retroviral regimens. Notch processing by γ-secretase results in cleavage of the notch intracellular domain that travels to the nucleus to regulate expression of genes such as vascular endothelial cell growth factor and NFκB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on γ-secretase substrate pathways in cerebral endothelial cell signaling have not been addressed, we sought to determine the effects of HIV PIs on Notch and amyloid precursor protein.ResultsExposure to reported physiological levels of Saquinavir, Indinavir, Nelfinavir and Ritonavir, significantly increased reactive oxygen species in cerebral endothelial cells, but had no effect on cell survival. Likewise, PIs decreased Notch 4-protein expression, but had no effect on Notch 1 or amyloid precursor protein expression. On the other hand, only Nelfinavir increased significantly Notch 4 processing, Notch4 intracellular domain nuclear localization and the expression of notch intracellular domain targets NFκB and matrix metalloproteinase 2. Pre-treatment with the antioxidant Vitamin E prevented PI-induced reactive oxygen species generation and partially prevented Nelfinavir-induced changes in both Notch 4 processing, and cellular localization patterns. Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity.ConclusionNelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFκB and matrix metalloproteinase 2 in cerebral endothelial cells.
Highlights
Aspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, specific mechanisms are poorly understood
HIV PIs induce the generation of reactive oxygen species (ROS) in CEC Reported plasma concentrations of PIs do not cause significant CEC death in vitro, studies show that PI exposure compromises cell fitness and signaling by inducing oxidative stress in non-cerebral endothelial cells [5,6]
1 h pre-treatment with the antioxidant, Vitamin E, prevented the generation of PI-induced ROS in both the 1 h and 48 h treated cells (p ≤ 0.001 and p ≤ 0.005, respectively) (Figure 2). These results show that the PIs tested induce significant levels of ROS in CEC, but Vitamin E pre-treatment prevents PI-induced ROS generation
Summary
Aspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, specific mechanisms are poorly understood. Protease inhibitors (PIs) used to treat HIV belong to an important group of drugs reported to influence significantly angiogenic capacity and endothelial cell functioning [4]. Adverse effects on non-cerebral endothelial cells by HIV PIs are well documented, the mechanisms responsible for dysregulation are poorly understood [4]. These aspartyl PIs, Nelfinavir (NFV), have been implicated in disruption of the Notch pathway in the HIV-related neoplasm, Kaposi's sarcoma [7]. Constant exposure of CEC to PIs circulating in the blood stream in the HIV patient likely affects normal CEC signaling pathways [8,9], such as Notch since its signaling is dependent on protease activity to maintain cell fitness [10,11]
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