Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir.
BACKGROUND
Aspartyl 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 gamma-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 NFkappaB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on gamma-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.


RESULTS
Exposure 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 NFkappaB 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.


CONCLUSIONS
Nelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFkappaB and matrix metalloproteinase 2 in cerebral endothelial cells.
Grigorian
Aline
A
Department of Pathology, University of California San Diego, La Jolla, USA. grigoria@sunstroke.sdsu.edu
Hurford
Rosemary
R
Chao
Ying
Y
Patrick
Christina
C
Langford
T Dianne
TD
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000166:cytology; D002550:Cerebral Veins / Q000166:cytology; D042783:Endothelial Cells / Q000187:drug effects*; D005625:Frontal Lobe / Q000098:blood supply / Q000166:cytology; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D019888:Nelfinavir / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000187:drug effects* / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D051881:Receptor, Notch1 / Q000378:metabolism; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000187:drug effects* / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D019438:Ritonavir / Q000494:pharmacology; D019258:Saquinavir / Q000494:pharmacology; D014810:Vitamin E / Q000494:pharmacology
C514503:GSKIP protein, human; D017320:HIV Protease Inhibitors; C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D051881:Receptor, Notch1; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D012097:Repressor Proteins; D014810:Vitamin E; D019469:Indinavir; D016282:Aspartic Acid Endopeptidases; D019888:Nelfinavir; D019258:Saquinavir; D019438:Ritonavir