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Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir.

Grigorian A, Hurford R, Chao Y, Patrick C, Langford TD - BMC Neurosci (2008)

Bottom Line: 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.Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of California San Diego, La Jolla, USA. grigoria@sunstroke.sdsu.edu

ABSTRACT

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.

Conclusion: 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.

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Related in: MedlinePlus

Nelfinavir increases NICD nuclear localization. CEC exposed to saquinavir (SQV), indinavir (INV), nelfinavir (NFV), and ritonavir (RTV) for 48 h were assessed for nuclear localization of NICD (green) and double labeled with actin (red). The percentage of cells positive for NICD nuclear localization was calculated by counting at least 10 random fields of view and at least 200 cells per condition. 60× magnification. *p ≤ 0.001 by one way ANOVA with Dunnett's post hoc tests when compared to control. **p ≤ 0.001 by one way ANOVA with Tukey-Kramer post hoc tests when compared to NFV treated cells.
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Figure 8: Nelfinavir increases NICD nuclear localization. CEC exposed to saquinavir (SQV), indinavir (INV), nelfinavir (NFV), and ritonavir (RTV) for 48 h were assessed for nuclear localization of NICD (green) and double labeled with actin (red). The percentage of cells positive for NICD nuclear localization was calculated by counting at least 10 random fields of view and at least 200 cells per condition. 60× magnification. *p ≤ 0.001 by one way ANOVA with Dunnett's post hoc tests when compared to control. **p ≤ 0.001 by one way ANOVA with Tukey-Kramer post hoc tests when compared to NFV treated cells.

Mentions: CEC untreated, and treated with PIs and/or Vitamin E for 48 h were assessed for both cytosolic and nuclear NICD immunoreactivity. Nuclear localization of NICD was detected in 21% of untreated CEC, 22% with SQV, 19% with INV and 26% with RTV (Figure 8). However, NFV-treated CEC displayed the highest percentage (34%) of cells positive for NICD nuclear localization (p ≤ 0.001) (Figure 8B). On the other hand, Vitamin E treatment alone resulted in decreased NICD nuclear localization (15%), compared to control. As expected, Vitamin E pre-treatment blocked the dramatic increase in nuclear NICD caused by NFV, dropping the percent cells positive down to 15.5% (Figure 8B). These results indicate that the NFV-induced increases in levels of NICD (Figure 3C, D, 4B) are accompanied by increased NICD localization to the nucleus and that Vitamin E pre-treatment prevents NFV-induced increased nuclear localization of NICD (Figure 8B).


Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir.

Grigorian A, Hurford R, Chao Y, Patrick C, Langford TD - BMC Neurosci (2008)

Nelfinavir increases NICD nuclear localization. CEC exposed to saquinavir (SQV), indinavir (INV), nelfinavir (NFV), and ritonavir (RTV) for 48 h were assessed for nuclear localization of NICD (green) and double labeled with actin (red). The percentage of cells positive for NICD nuclear localization was calculated by counting at least 10 random fields of view and at least 200 cells per condition. 60× magnification. *p ≤ 0.001 by one way ANOVA with Dunnett's post hoc tests when compared to control. **p ≤ 0.001 by one way ANOVA with Tukey-Kramer post hoc tests when compared to NFV treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268698&req=5

Figure 8: Nelfinavir increases NICD nuclear localization. CEC exposed to saquinavir (SQV), indinavir (INV), nelfinavir (NFV), and ritonavir (RTV) for 48 h were assessed for nuclear localization of NICD (green) and double labeled with actin (red). The percentage of cells positive for NICD nuclear localization was calculated by counting at least 10 random fields of view and at least 200 cells per condition. 60× magnification. *p ≤ 0.001 by one way ANOVA with Dunnett's post hoc tests when compared to control. **p ≤ 0.001 by one way ANOVA with Tukey-Kramer post hoc tests when compared to NFV treated cells.
Mentions: CEC untreated, and treated with PIs and/or Vitamin E for 48 h were assessed for both cytosolic and nuclear NICD immunoreactivity. Nuclear localization of NICD was detected in 21% of untreated CEC, 22% with SQV, 19% with INV and 26% with RTV (Figure 8). However, NFV-treated CEC displayed the highest percentage (34%) of cells positive for NICD nuclear localization (p ≤ 0.001) (Figure 8B). On the other hand, Vitamin E treatment alone resulted in decreased NICD nuclear localization (15%), compared to control. As expected, Vitamin E pre-treatment blocked the dramatic increase in nuclear NICD caused by NFV, dropping the percent cells positive down to 15.5% (Figure 8B). These results indicate that the NFV-induced increases in levels of NICD (Figure 3C, D, 4B) are accompanied by increased NICD localization to the nucleus and that Vitamin E pre-treatment prevents NFV-induced increased nuclear localization of NICD (Figure 8B).

Bottom Line: 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.Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of California San Diego, La Jolla, USA. grigoria@sunstroke.sdsu.edu

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus